Secondary Wall Forming Genes From Maize and Uses Thereof

ABSTRACT

The present invention provides polynucleotides and related polypeptides of the class of genes involved in maize secondary wall (ZmSCW) formation. The invention provides genomic sequence for the ZmSCW genes. ZmSCW are responsible for controlling plant growth, secondary cell wall development and yield in crop plants.

CROSS REFERENCE

This utility application claims the benefit U.S. Provisional ApplicationNo. 60/944,117, filed Jun. 15, 2007 which is incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates generally to the field of molecular biology.

BACKGROUND OF THE INVENTION

Harvest index, ratio of grain to total aboveground biomass, has remainednearly constant around 50% in maize over the past 100 years. (Sinclair,(1998) “Historical changes in harvest index and crop nitrogenaccumulation”; Crop Science 38:638-643); (Tollenaar and Wu. (1999)“Yield improvement in temperate maize is attributable to greater stresstolerance”; Crop Science 39:1597-1604). Thus, the quadrupling of grainyield over the last 50-60 years has resulted from an increase in totalbiomass production per unit land area, which has been accomplished byincreased planting density (Duvick and Cassman, (1999) “Post-greenrevolution trends in yield potential of temperate maize in thenorth-central United States”; Crop Science 39:1622-1630). Selection forhigher grain yield under increasing planting densities has led to asignificant architectural change in plant structure, that of relativelyerect and narrow leaves to minimize shading. An undesirable consequenceof denser planting has been the increased frequency of stalk lodging.The relationship between planting density and biomass productiondeviates significantly from linearity as the optimal density isapproached for maximal biomass yield per unit land area. This isreflected in a proportionately greater reduction in the individual plantbiomass, which manifests in the form of weaker stalks and henceincreased lodging.

Cellulose in a unit length of the maize stalk was found to be the bestindicator of mechanical strength (Appenzeller, et al., (2004) “Cellulosesynthesis in maize: isolation and expression analysis of the cellulosesynthase (CesA) gene family”; Cellulose 11:287-299; Ching, et al.,(2006) “Brittle stalk 2 encodes a putativeglycosylphosphatidylinositol-anchored protein that affects mechanicalstrength of maize tissues by altering the composition and structure ofsecondary cell walls”; Planta 224:1174-1184). Cellulose constitutesapproximately 50% of the stalk dry matter at maturity (Dhugga,unpublished). Whereas the concentration of cellulose in the maize stalkwall can vary considerably in the germplasm that for lignin isessentially invariable (Dhugga, unpublished). This implies that theconcentration of cellulose varies at the expense of other cell wallcomponents such as hemicellulose and soluble components. Increasingcellulose concentration in the dry matter should allow improving harvestindex without adversely affecting stalk mechanical strength. Thus themost preferred means of improving stalk strength are through increasedcellulose concentration and secondary wall content.

This invention pertains to a set of maize genes involved in cell wallformation, in particular secondary cell wall formation (SCW). Cellulosemay constitute up to 60% of the secondary cell wall of plants such asmaize. The genes that are subject of this invention are revealed to beassociated with secondary cell wall formation based on the strongcorrelation of their expression patterns with those of ZmCesA10,ZmCesA11, ZmCesA12, ZmCesA13, and Bk2 genes that had previously beenshown to be involved in secondary cell wall formation (Appenzeller, etal., (2004) “Cellulose synthesis in maize: isolation and expressionanalysis of the cellulose synthase (CesA) gene family”; Cellulose11:287-299; Ching, et al., (2006) “Brittle stalk 2 encodes a putativeglycosylphosphatidylinositol-anchored protein that affects mechanicalstrength of maize tissues by altering the composition and structure ofsecondary cell walls”; Planta 224:1174-1184; Dhugga, unpublished). Manyof the genes in this invention are not known to be associated with cellwall formation outside of these proprietary analyses. These genes couldbe used to enhance crop plant performance and value in several areasincluding: 1) plant standability, harvest index, and yield potential; 2)plant dry matter as a feedstock for ethanol or for other renewablebioproducts; and 3) silage.

In addition to its role as the primary determinant of tissue strength, atrait that is of significant interest in agriculture, celluloseconstitutes the most abundant renewable energy resource on Earth.Approximately 275 million metric tons of stover is produced just frommaize in the USA every year. About two-thirds of stover couldpotentially be utilized for ethanol, butanol and other fuels orbioproducts from some corn-growing regions (Graham, et al., (2007)“Current and potential U.S. corn stover supplies”; Agronomy Journal99:1-11). The worldwide production of lignocellulosic wastes from cerealstover and straw is estimated to be ˜3 billion tons per year (Kuhad andSingh (1993) “Lignocellulose biotechnology: Current and futureprospects”; Critic. Rev. Biotechnol. 13:151-172). Secondary wallaccounts for a great majority of the vegetative biomass in theterrestrial vegetation and is thus a suitable target for manipulation toimprove the amount and quality of biomass for energy production.Alteration of secondary wall for improved silage quality may beaccomplished by altering lignin concentration. Both the type and amountof lignin have long been known to affect silage digestibility. Lignin isalso an impediment in the digestion of cell wall polysaccharides forethanol production. Several of the genes in our list expand the numberof candidates we can use to alter the composition of cell wall forimproved silage quality.

This invention provides solutions to agronomic problems in at leastthree areas: 1) plant standability, harvest index, and yield potential;2) plant dry matter as a feedstock for ethanol or for other renewablebioproducts; and 3) silage digestibility.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods for controlling plant growth and secondary cellwall formation for increasing yield in a plant are provided. Thecompositions include SCW sequences from maize. Compositions of theinvention comprise amino acid sequences and nucleotide sequencesselected from SEQ ID NOS: 1-456 as well as variants and fragmentsthereof.

Polynucleotides encoding the SCW sequences are provided in DNAconstructs for expression in a plant of interest. Expression cassettes,plants, plant cells, plant parts, and seeds comprising the sequences ofthe invention are further provided. In specific embodiments, thepolynucleotide is operably linked to a constitutive promoter.

Methods for modulating the level of an SCW sequence in a plant or plantpart are provided. The methods comprise introducing into a plant orplant part a heterologous polynucleotide comprising an SCW sequence ofthe invention. The level of SCW polypeptide can be increased ordecreased. Such method can be used to increase the yield in plants; inone embodiment, the method is used to increase grain yield in cereals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Relative contribution of maize stalk rind and inner tissue todifferent stalk characteristics and mechanical strength.

FIG. 2: Cellulose concentration in dry matter of maize hybrids. Datawere collected from two plants derived from each of the three fieldreplications. Cellulose was determined by Updegraff method (Updegraff(1969) “Semimicro determination of cellulose in biological materials”;Anal. Biochem. 32:120-124) on powdered dry matter obtained from thethird internode below the ear at maturity.

FIG. 3: Transcript profile across twelve maize tissues for 4 referencesecondary cell wall genes and 38 discovered genes. Messenger RNAprofiles of the four Reference secondary cell wall genes (Bk2, CesA10,CesA11, CesA12) and the 38 discovered genes that have correlatedexpression patterns. The tissue expression levels represent mean (andSE) values for the MPSS 17-mer tag sets representing the genes in forthe reference and discovered sets. The overall gene expression patternis very similar between the sets, showing peak expression in stalks,slighter expression in roots and leaves, and little expression in othertissues, in particular apical meristem, which lacks secondary cellwalls. The mean expression magnitudes are less important than thecorrespondence of the tissue expression patterns, as transcript and taglevels between genes will vary naturally and for technical reasons.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting. The following ispresented by way of illustration and is not intended to limit the scopeof the invention.

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of botany, microbiology, tissueculture, molecular biology, chemistry, biochemistry and recombinant DNAtechnology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, e.g., Langenheim and Thimann,BOTANY: PLANT BIOLOGY AND ITS RELATION TO HUMAN AFFAIRS, John Wiley(1982); CELL CULTURE AND SOMATIC CELL GENETICS OF PLANTS, vol. 1, Vasil,ed. (1984); Stanier, et al., THE MICROBIAL WORLD, 5^(th) ed.,Prentice-Hall (1986); Dhringra and Sinclair, BASIC PLANT PATHOLOGYMETHODS, CRC Press (1985); Maniatis, et al., MOLECULAR CLONING: ALABORATORY MANUAL (1982); DNA CLONING, vols. I and II, Glover, ed.(1985); OLIGONUCLEOTIDE SYNTHESIS, Gait, ed. (1984); NUCLEIC ACIDHYBRIDIZATION, Hames and Higgins, eds. (1984); and the series METHODS INENZYMOLOGY, Colowick and Kaplan, eds, Academic Press, Inc., San Diego,Calif.

Units, prefixes, and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range. Amino acids may be referred to hereinby either their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The terms defined below are more fullydefined by reference to the specification as a whole.

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

By “microbe” is meant any microorganism (including both eukaryotic andprokaryotic microorganisms), such as fungi, yeast, bacteria,actinomycetes, algae and protozoa, as well as other unicellularstructures.

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerase chain reaction (PCR)system, ligase chain reaction (LCR) system, nucleic acid sequence basedamplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicasesystems, transcription-based amplification system (TAS), and stranddisplacement amplification (SDA). See, e.g., DIAGNOSTIC MOLECULARMICROBIOLOGY: PRINCIPLES AND APPLICATIONS, Persing, et al., eds.,American Society for Microbiology, Washington, D.C. (1993). The productof amplification is termed an amplicon.

The term “conservatively modified variants” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refer to those nucleic acidsthat encode identical or conservatively modified variants of the aminoacid sequences. Because of the degeneracy of the genetic code, a largenumber of functionally identical nucleic acids encode any given protein.For instance, the codons GCA, GCC, GCG and GCU all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations” and represent one species ofconservatively modified variation. Every nucleic acid sequence hereinthat encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of ordinary skill will recognize thateach codon in a nucleic acid (except AUG, which is ordinarily the onlycodon for methionine; one exception is Micrococcus rubens, for which GTGis the methionine codon (Ishizuka, et al., (1993) J. Gen. Microbiol.139:425-32) can be modified to yield a functionally identical molecule.Accordingly, each silent variation of a nucleic acid, which encodes apolypeptide of the present invention, is implicit in each describedpolypeptide sequence and incorporated herein by reference.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” when the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Thus, any number of amino acid residues selected from the group ofintegers consisting of from 1 to 15 can be so altered. Thus, forexample, 1, 2, 3, 4, 5, 7 or 10 alterations can be made. Conservativelymodified variants typically provide similar biological activity as theunmodified polypeptide sequence from which they are derived. Forexample, substrate specificity, enzyme activity, or ligand/receptorbinding is generally at least 30%, 40%, 50%, 60%, 70%, 80% or 90%,preferably 60-90% of the native protein for it's native substrate.Conservative substitution tables providing functionally similar aminoacids are well known in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

See also, Creighton, PROTEINS, W.H. Freeman and Co. (1984).

As used herein, “consisting essentially of” means the inclusion ofadditional sequences to an object polynucleotide where the additionalsequences do not selectively hybridize, under stringent hybridizationconditions, to the same cDNA as the polynucleotide and where thehybridization conditions include a wash step in 0.1×SSC and 0.1% sodiumdodecyl sulfate at 65° C.

By “encoding” or “encoded,” with respect to a specified nucleic acid, ismeant comprising the information for translation into the specifiedprotein. A nucleic acid encoding a protein may comprise non-translatedsequences (e.g., introns) within translated regions of the nucleic acid,or may lack such intervening non-translated sequences (e.g., as incDNA). The information by which a protein is encoded is specified by theuse of codons. Typically, the amino acid sequence is encoded by thenucleic acid using the “universal” genetic code. However, variants ofthe universal code, such as is present in some plant, animal, and fungalmitochondria, the bacterium Mycoplasma capricolumn (Yamao, et al.,(1985) Proc. Natl. Acad. Sci. USA 82:2306-2309), or the ciliateMacronucleus, may be used when the nucleic acid is expressed using theseorganisms.

When the nucleic acid is prepared or altered synthetically, advantagecan be taken of known codon preferences of the intended host where thenucleic acid is to be expressed. For example, although nucleic acidsequences of the present invention may be expressed in bothmonocotyledonous and dicotyledonous plant species, sequences can bemodified to account for the specific codon preferences and GC contentpreferences of monocotyledonous plants or dicotyledonous plants as thesepreferences have been shown to differ (Murray, et al., (1989) NucleicAcids Res. 17:477-98 and herein incorporated by reference). Thus, themaize preferred codon for a particular amino acid might be derived fromknown gene sequences from maize. Maize codon usage for 28 genes frommaize plants is listed in Table 4 of Murray, et al., supra.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived or, if from the same species, one or both are substantiallymodified from their original form. A heterologous protein may originatefrom a foreign species or, if from the same species, is substantiallymodified from its original form by deliberate human intervention.

By “host cell” is meant a cell, which contains a vector and supports thereplication and/or expression of the expression vector. Host cells maybe prokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, plant, amphibian, or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledonous plant cells, including but notlimited to maize, sorghum, sunflower, soybean, wheat, alfalfa, rice,cotton, canola, barley, millet, and tomato. A particularly preferredmonocotyledonous host cell is a maize host cell.

The term “hybridization complex” includes reference to a duplex nucleicacid structure formed by two single-stranded nucleic acid sequencesselectively hybridized with each other.

The term “introduced” in the context of inserting a nucleic acid into acell, means “transfection” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

The terms “isolated” refers to material, such as a nucleic acid or aprotein, which is substantially or essentially free from componentswhich normally accompany or interact with it as found in its naturallyoccurring environment. The isolated material optionally comprisesmaterial not found with the material in its natural environment. Nucleicacids, which are “isolated”, as defined herein, are also referred to as“heterologous” nucleic acids. Unless otherwise stated, the term “SCWnucleic acid” means a nucleic acid comprising a polynucleotide (“SCWpolynucleotide”) encoding a SCW polypeptide.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides (e.g., peptide nucleic acids).

By “nucleic acid library” is meant a collection of isolated DNA or RNAmolecules, which comprise and substantially represent the entiretranscribed fraction of a genome of a specified organism. Constructionof exemplary nucleic acid libraries, such as genomic and cDNA libraries,is taught in standard molecular biology references such as Berger andKimmel, GUIDE TO MOLECULAR CLONING TECHNIQUES, from the series METHODSIN ENZYMOLOGY, vol. 152, Academic Press, Inc., San Diego, Calif. (1987);Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2^(nd) ed.,vols. 1-3 (1989); and CURRENT PROTOCOLS 1N MOLECULAR BIOLOGY, Ausubel,et al., eds, Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc. (1994Supplement).

As used herein “operably linked” includes reference to a functionallinkage between a first sequence, such as a promoter and a secondsequence, wherein the promoter sequence initiates and mediatestranscription of the DNA sequence corresponding to the second sequence.Generally, operably linked means that the nucleic acid sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in the same reading frame.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cellsand progeny of same. Plant cell, as used herein includes, withoutlimitation, seeds suspension cultures, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,and microspores. The class of plants, which can be used in the methodsof the invention, is generally as broad as the class of higher plantsamenable to transformation techniques, including both monocotyledonousand dicotyledonous plants including species from the genera: Cucurbita,Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium,Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus,Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura,Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis,Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum,Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium, and Triticum.A particularly preferred plant is Zea mays.

As used herein, “yield” includes reference to bushels per acre of agrain crop at harvest, as adjusted for grain moisture (15% typically).Grain moisture is measured in the grain at harvest. The adjusted testweight of grain is determined to be the weight in pounds per bushel,adjusted for grain moisture level at harvest.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide, or analogs thereof thathave the essential nature of a natural ribonucleotide in that theyhybridize, under stringent hybridization conditions, to substantiallythe same nucleotide sequence as naturally occurring nucleotides and/orallow translation into the same amino acid(s) as the naturally occurringnucleotide(s). A polynucleotide can be full-length or a subsequence of anative or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including inter alia, simple andcomplex cells.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Exemplary plant promoters include, but are not limited to, thosethat are obtained from plants, plant viruses, and bacteria whichcomprise genes expressed in plant cells such Agrobacterium or Rhizobium.Examples are promoters that preferentially initiate transcription incertain tissues, such as leaves, roots, seeds, fibres, xylem vessels,tracheids, or sclerenchyma. Such promoters are referred to as “tissuepreferred.” A “cell type” specific promoter primarily drives expressionin certain cell types in one or more organs, for example, vascular cellsin roots or leaves. An “inducible” or “regulatable” promoter is apromoter, which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light. Anothertype of promoter is a developmentally regulated promoter, for example, apromoter that drives expression during pollen development. Tissuepreferred, cell type specific, developmentally regulated, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter, which is active under mostenvironmental conditions.

The term “SCW polypeptide” refers to one or more amino acid sequences.The term is also inclusive of fragments, variants, homologs, alleles orprecursors (e.g., preproproteins or proproteins) thereof. A “SCWprotein” comprises a SCW polypeptide. Unless otherwise stated, the term“SCW nucleic acid” means a nucleic acid comprising a polynucleotide(“SCW polynucleotide”) encoding a SCW polypeptide.

As used herein “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found in identicalform within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under expressed ornot expressed at all as a result of deliberate human intervention. Theterm “recombinant” as used herein does not encompass the alteration ofthe cell or vector by naturally occurring events (e.g., spontaneousmutation, natural transformation/transduction/transposition) such asthose occurring without deliberate human intervention.

As used herein, a “recombinant expression cassette” is a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements, which permit transcription of aparticular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed, and apromoter.

The term “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass known analogs of natural amino acids that canfunction in a similar manner as naturally occurring amino acids.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 40% sequence identity, preferably 60-90% sequenceidentity, and most preferably 100% sequence identity (i.e.,complementary) with each other.

The terms “stringent conditions” or “stringent hybridization conditions”include reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than other sequences(e.g., at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified which can be up to 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Optimally, the probe is approximately 500 nucleotides inlength, but can vary greatly in length from less than 500 nucleotides toequal to the entire length of the target sequence.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide or Denhardt's.Exemplary low stringency conditions include hybridization with a buffersolution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 Mtrisodium citrate) at 50 to 55° C. Exemplary moderate stringencyconditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1%SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplaryhigh stringency conditions include hybridization in 50% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-84:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the thermalmelting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than thethermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY—HYBRIDIZATION WITH NUCLEIC ACID PROBES, part I, chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” Elsevier, N.Y. (1993); and CURRENT PROTOCOLS INMOLECULAR BIOLOGY, chapter 2, Ausubel, et al., eds, Greene Publishingand Wiley-Interscience, New York (1995). Unless otherwise stated, in thepresent application high stringency is defined as hybridization in4×SSC, 5×Denhardt's (5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovineserum albumin in 500 ml of water), 0.1 mg/ml boiled salmon sperm DNA,and 25 mM Na phosphate at 65° C., and a wash in 0.1×SSC, 0.1% SDS at 65°C.

As used herein, “transgenic plant” includes reference to a plant, whichcomprises within its genome a heterologous polynucleotide. Generally,the heterologous polynucleotide is stably integrated within the genomesuch that the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant expression cassette. “Transgenic” is usedherein to include any cell, cell line, callus, tissue, plant part orplant, the genotype of which has been altered by the presence ofheterologous nucleic acid including those transgenics initially soaltered as well as those created by sexual crosses or asexualpropagation from the initial transgenic. The term “transgenic” as usedherein does not encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally occurring events such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition, or spontaneous mutation.

As used herein, “vector” includes reference to a nucleic acid used intransfection of a host cell and into which can be inserted apolynucleotide. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides or polypeptides:(a) “reference sequence,” (b) “comparison window,” (c) “sequenceidentity,” (d) “percentage of sequence identity,” and (e) “substantialidentity.”

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

As used herein, “comparison window” means includes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence may be compared to a reference sequence andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. Generally, the comparison windowis at least 20 contiguous nucleotides in length, and optionally can be30, 40, 50, 100 or longer. Those of skill in the art understand that toavoid a high similarity to a reference sequence due to inclusion of gapsin the polynucleotide sequence a gap penalty is typically introduced andis subtracted from the number of matches.

Methods of alignment of nucleotide and amino acid sequences forcomparison are well known in the art. The local homology algorithm(BESTFIT) of Smith and Waterman, (1981) Adv. Appl. Math 2:482, mayconduct optimal alignment of sequences for comparison; by the homologyalignment algorithm (GAP) of Needleman and Wunsch, (1970) J. Mol. Biol.48:443-53; by the search for similarity method (Tfasta and Fasta) ofPearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444; bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Version 8 (available from Genetics ComputerGroup (GCG® programs (Accelrys, Inc., San Diego, Calif.)). The CLUSTALprogram is well described by Higgins and Sharp, (1988) Gene 73:237-44;Higgins and Sharp, (1989) CABIOS 5:151-3; Corpet, et al., (1988) NucleicAcids Res. 16:10881-90; Huang, et al., (1992) Computer Applications inthe Biosciences 8:155-65, and Pearson, et al., (1994) Meth. Mol. Biol.24:307-31. The preferred program to use for optimal global alignment ofmultiple sequences is PileUp (Feng and Doolittle, (1987) J. Mol. Evol.,25:351-60 which is similar to the method described by Higgins and Sharp,(1989) CABIOS 5:151-53 and hereby incorporated by reference). The BLASTfamily of programs which can be used for database similarity searchesincludes: BLASTN for nucleotide query sequences against nucleotidedatabase sequences; BLASTX for nucleotide query sequences againstprotein database sequences; BLASTP for protein query sequences againstprotein database sequences; TBLASTN for protein query sequences againstnucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences. See, CURRENT PROTOCOLSIN MOLECULAR BIOLOGY, Chapter 19, Ausubel, et al., eds., GreenePublishing and Wiley-Interscience, New York (1995).

GAP uses the algorithm of Needleman and Wunsch, supra, to find thealignment of two complete sequences that maximizes the number of matchesand minimizes the number of gaps. GAP considers all possible alignmentsand gap positions and creates the alignment with the largest number ofmatched bases and the fewest gaps. It allows for the provision of a gapcreation penalty and a gap extension penalty in units of matched bases.GAP must make a profit of gap creation penalty number of matches foreach gap it inserts. If a gap extension penalty greater than zero ischosen, GAP must, in addition, make a profit for each gap inserted ofthe length of the gap times the gap extension penalty. Default gapcreation penalty values and gap extension penalty values in Version 10of the Wisconsin Genetics Software Package are 8 and 2, respectively.The gap creation and gap extension penalties can be expressed as aninteger selected from the group of integers consisting of from 0 to 100.Thus, for example, the gap creation and gap extension penalties can be0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl.Acad. Sci. USA 89:10915).

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the BLAST 2.0 suite of programsusing default parameters (Altschul, et al., (1997) Nucleic Acids Res.25:3389-402).

As those of ordinary skill in the art will understand, BLAST searchesassume that proteins can be modeled as random sequences. However, manyreal proteins comprise regions of nonrandom sequences, which may behomopolymeric tracts, short-period repeats, or regions enriched in oneor more amino acids. Such low-complexity regions may be aligned betweenunrelated proteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs can be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Claverie andStates, (1993) Comput. Chem. 17:191-201) low-complexity filters can beemployed alone or in combination.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences includes reference to the residuesin the two sequences, which are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences, which differ by suchconservative substitutions, are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., according to the algorithm of Meyersand Miller, (1988) Computer Applic. Biol. Sci. 4:11-17, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has between 50-100% sequenceidentity, preferably at least 50% sequence identity, preferably at least60% sequence identity, preferably at least 70%, more preferably at least80%, more preferably at least 90%, and most preferably at least 95%,compared to a reference sequence using one of the alignment programsdescribed using standard parameters. One of skill will recognize thatthese values can be appropriately adjusted to determine correspondingidentity of proteins encoded by two nucleotide sequences by taking intoaccount codon degeneracy, amino acid similarity, reading framepositioning and the like. Substantial identity of amino acid sequencesfor these purposes normally means sequence identity of between 55-100%,preferably at least 55%, preferably at least 60%, more preferably atleast 70%, 80%, 90% and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.The degeneracy of the genetic code allows for many amino acidssubstitutions that lead to variety in the nucleotide sequence that codefor the same amino acid, hence it is possible that the DNA sequencecould code for the same polypeptide but not hybridize to each otherunder stringent conditions. This may occur, e.g., when a copy of anucleic acid is created using the maximum codon degeneracy permitted bythe genetic code. One indication that two nucleic acid sequences aresubstantially identical is that the polypeptide, which the first nucleicacid encodes, is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

The terms “substantial identity” in the context of a peptide indicatesthat a peptide comprises a sequence with between 55-100% sequenceidentity to a reference sequence preferably at least 55% sequenceidentity, preferably 60% preferably 70%, more preferably 80%, mostpreferably at least 90% or 95% sequence identity to the referencesequence over a specified comparison window. Preferably, optimalalignment is conducted using the homology alignment algorithm ofNeedleman and Wunsch, supra. An indication that two peptide sequencesare substantially identical is that one peptide is immunologicallyreactive with antibodies raised against the second peptide. Thus, apeptide is substantially identical to a second peptide, for example,where the two peptides differ only by a conservative substitution. Inaddition, a peptide can be substantially identical to a second peptidewhen they differ by a non-conservative change if the epitope that theantibody recognizes is substantially identical. Peptides, which are“substantially similar” share sequences as, noted above except thatresidue positions, which are not identical, may differ by conservativeamino acid changes.

The invention discloses SCW polynucleotides and polypeptides. The novelnucleotides and proteins of the invention have an expression patternwhich indicates that they alter cell wall formation and thus play animportant role in plant development. The polynucleotides are expressedin various plant tissues. The polynucleotides and polypeptides thusprovide an opportunity to manipulate plant development to alter seed andvegetative tissue development, timing or composition. This may be usedto create a sterile plant, a seedless plant or a plant with alteredendosperm composition.

Nucleic Acids

The present invention provides, inter alia, isolated nucleic acids ofRNA, DNA, and analogs and/or chimeras thereof, comprising a SCWpolynucleotide.

The present invention also includes polynucleotides optimized forexpression in different organisms. For example, for expression of thepolynucleotide in a maize plant, the sequence can be altered to accountfor specific codon preferences and to alter GC content as according toMurray, et al, supra. Maize codon usage for 28 genes from maize plantsis listed in Table 4 of Murray, et al., supra.

The SCW nucleic acids of the present invention comprise isolated SCWpolynucleotides which are inclusive of:

-   -   (a) a polynucleotide encoding a SCW polypeptide and        conservatively modified and polymorphic variants thereof;    -   (b) a polynucleotide having at least 70% sequence identity with        polynucleotides of (a) or (b);    -   (c) complementary sequences of polynucleotides of (a) or (b).

The following table, Table 1, lists the specific identities of the Zeamays sequences disclosed herein. An additional table, Table 2, lists thespecific identities of the SCW sequence orthologs in Oryza sativa (Os),Arabidopsis thaliana (At), Populus trichocarpa (Pt), Medicago truncatula(Mt), and Sorghum bicolor (Sb) disclosed herein.

TABLE 1 SCW group Maize Sequence Identity SEQ ID NO: SCW01Polynucleotide- ORF SEQ ID NO: 1 Polypeptide SEQ ID NO: 2 Polynucleotidetranscript SEQ ID NO: 77 Promoter SEQ ID NO: 425 SCW04 Polynucleotide-ORF SEQ ID NO: 3 Polypeptide SEQ ID NO: 4 Polynucleotide transcript SEQID NO: 78 SCW05 Polynucleotide- ORF SEQ ID NO: 5 Polypeptide SEQ ID NO:6 Polynucleotide transcript SEQ ID NO: 79 Promoter SEQ ID NO: 426 SCW06Polynucleotide- ORF SEQ ID NO: 7 Polypeptide SEQ ID NO: 8 Polynucleotidetranscript SEQ ID NO: 80 Promoter SEQ ID NO: 427 SCW08 Polynucleotide-ORF SEQ ID NO: 9 Polypeptide SEQ ID NO: 10 Polynucleotide transcript SEQID NO: 81 SCW09 Polynucleotide- ORF SEQ ID NO: 11 Polypeptide SEQ ID NO:12 Polynucleotide transcript SEQ ID NO: 82 SCW10 Polynucleotide- ORF SEQID NO: 13 Polypeptide SEQ ID NO: 14 Polynucleotide transcript SEQ ID NO:83 Promoter SEQ ID NO: 428 SCW11a Polynucleotide- ORF SEQ ID NO: 15Polypeptide SEQ ID NO: 16 Polynucleotide transcript SEQ ID NO: 84Promoter SEQ ID NO: 429 SCW11b Polynucleotide- ORF SEQ ID NO: 17Polypeptide SEQ ID NO: 18 Polynucleotide transcript SEQ ID NO: 85Promoter SEQ ID NO: 430 SCW13 Polynucleotide- ORF SEQ ID NO: 19Polypeptide SEQ ID NO: 20 Polynucleotide transcript SEQ ID NO: 86Promoter SEQ ID NO: 431 SCW16 Polynucleotide- ORF SEQ ID NO: 21Polypeptide SEQ ID NO: 22 Polynucleotide transcript SEQ ID NO: 87Promoter SEQ ID NO: 432 SCW17 Polynucleotide- ORF SEQ ID NO: 23Polypeptide SEQ ID NO: 24 Polynucleotide transcript SEQ ID NO: 88Promoter SEQ ID NO: 433 SCW21 Polynucleotide- ORF SEQ ID NO: 25Polypeptide SEQ ID NO: 26 Polynucleotide transcript SEQ ID NO: 89Promoter SEQ ID NO: 434 SCW22 Polynucleotide- ORF SEQ ID NO: 27Polypeptide SEQ ID NO: 28 Polynucleotide transcript SEQ ID NO: 90Promoter SEQ ID NO: 435 SCW23 Polynucleotide- ORF SEQ ID NO: 29Polypeptide SEQ ID NO: 30 Polynucleotide transcript SEQ ID NO: 91Promoter SEQ ID NO: 436 SCW26 Polynucleotide- ORF SEQ ID NO: 31Polypeptide SEQ ID NO: 32 Polynucleotide transcript SEQ ID NO: 92 SCW28Polynucleotide- ORF SEQ ID NO: 33 Polypeptide SEQ ID NO: 34Polynucleotide transcript SEQ ID NO: 93 Promoter SEQ ID NO: 437 SCW32Polynucleotide- ORF SEQ ID NO: 35 Polypeptide SEQ ID NO: 36Polynucleotide transcript SEQ ID NO: 94 SCW34 Polynucleotide- ORF SEQ IDNO: 37 Polypeptide SEQ ID NO: 38 Polynucleotide transcript SEQ ID NO: 95Promoter SEQ ID NO: 438 SCW38 Polynucleotide- ORF SEQ ID NO: 39Polypeptide SEQ ID NO: 40 Polynucleotide transcript SEQ ID NO: 96Promoter SEQ ID NO: 439 SCW39 Polynucleotide- ORF SEQ ID NO: 41Polypeptide SEQ ID NO: 42 Polynucleotide transcript SEQ ID NO: 97Promoter SEQ ID NO: 440 SCW40 Polynucleotide- ORF SEQ ID NO: 43Polypeptide SEQ ID NO: 44 Polynucleotide transcript SEQ ID NO: 98Promoter SEQ ID NO: 441 SCW41 Polynucleotide- ORF SEQ ID NO: 45Polypeptide SEQ ID NO: 46 Polynucleotide transcript SEQ ID NO: 99Promoter SEQ ID NO: 442 SCW43 Polynucleotide- ORF SEQ ID NO: 47Polypeptide SEQ ID NO: 48 Polynucleotide transcript SEQ ID NO: 100Promoter SEQ ID NO: 443 SCW44 Polynucleotide- ORF SEQ ID NO: 49Polypeptide SEQ ID NO: 50 Polynucleotide transcript SEQ ID NO: 101Promoter SEQ ID NO: 444 SCW45 Polynucleotide- ORF SEQ ID NO: 51Polypeptide SEQ ID NO: 52 Polynucleotide transcript SEQ ID NO: 102Promoter SEQ ID NO: 445 SCW46 Polynucleotide- ORF SEQ ID NO: 53Polypeptide SEQ ID NO: 54 Polynucleotide transcript SEQ ID NO: 103Promoter SEQ ID NO: 446 SCW47 Polynucleotide- ORF SEQ ID NO: 55Polypeptide SEQ ID NO: 56 Polynucleotide transcript SEQ ID NO: 104Promoter SEQ ID NO: 447 SCW48 Polynucleotide- ORF SEQ ID NO: 57Polypeptide SEQ ID NO: 58 Polynucleotide transcript SEQ ID NO: 105Promoter SEQ ID NO: 448 SCW49 Polynucleotide- ORF SEQ ID NO: 59Polypeptide SEQ ID NO: 60 Polynucleotide transcript SEQ ID NO: 106Promoter SEQ ID NO: 449 SCW50 Polynucleotide- ORF SEQ ID NO: 61Polypeptide SEQ ID NO: 62 Polynucleotide transcript SEQ ID NO: 107Promoter SEQ ID NO: 450 SCW51 Polynucleotide- ORF SEQ ID NO: 63Polypeptide SEQ ID NO: 64 Polynucleotide transcript SEQ ID NO: 108 SCW53Polynucleotide- ORF SEQ ID NO: 65 Polypeptide SEQ ID NO: 66Polynucleotide transcript SEQ ID NO: 109 Promoter SEQ ID NO: 451 SCW54Polynucleotide- ORF SEQ ID NO: 67 Polypeptide SEQ ID NO: 68Polynucleotide transcript SEQ ID NO: 110 Promoter SEQ ID NO: 452 SCW55Polynucleotide- ORF SEQ ID NO: 69 Polypeptide SEQ ID NO: 70Polynucleotide transcript SEQ ID NO: 111 Promoter SEQ ID NO: 453 SCW56Polynucleotide- ORF SEQ ID NO: 71 Polypeptide SEQ ID NO: 72Polynucleotide transcript SEQ ID NO: 112 Promoter SEQ ID NO: 454 SCW57Polynucleotide- ORF SEQ ID NO: 73 Polypeptide SEQ ID NO: 74Polynucleotide transcript SEQ ID NO: 113 Promoter SEQ ID NO: 455 SCW58Polynucleotide- ORF SEQ ID NO: 75 Polypeptide SEQ ID NO: 76Polynucleotide transcript SEQ ID NO: 114 Promoter SEQ ID NO: 456

TABLE 2 SCW group for other species Identity SEQ ID NO: SCW01 At1Polypeptide SEQ ID NO: 115 At1 Polynucleotide transcript SEQ ID NO: 116At2 Polypeptide SEQ ID NO: 117 At2 Polynucleotide transcript SEQ ID NO:118 Os Polypeptide SEQ ID NO: 119 Os Polynucleotide transcript SEQ IDNO: 120 Pt1 Polypeptide SEQ ID NO: 121 Pt1 Polynucleotide transcript SEQID NO: 122 Pt2 Polypeptide SEQ ID NO: 123 Pt2 Polynucleotide transcriptSEQ ID NO: 124 SCW04 At1 Polypeptide SEQ ID NO: 125 At1 Polynucleotidetranscript SEQ ID NO: 126 At2 Polypeptide SEQ ID NO: 127 At2Polynucleotide transcript SEQ ID NO: 128 Mt Polypeptide SEQ ID NO: 129Mt Polynucleotide transcript SEQ ID NO: 130 Os Polypeptide SEQ ID NO:131 Os Polynucleotide transcript SEQ ID NO: 132 Pt1 Polypeptide SEQ IDNO: 133 Pt1 Polynucleotide transcript SEQ ID NO: 134 Pt2 Polypeptide SEQID NO: 135 Pt2 Polynucleotide transcript SEQ ID NO: 136 SCW05 OsPolypeptide SEQ ID NO: 137 Os Polynucleotide transcript SEQ ID NO: 138SCW06 At1 Polypeptide SEQ ID NO: 139 At1 Polynucleotide transcript SEQID NO: 140 At2 Polypeptide SEQ ID NO: 141 At2 Polynucleotide transcriptSEQ ID NO: 142 Mt Polypeptide SEQ ID NO: 143 Mt Polynucleotidetranscript SEQ ID NO: 144 Os Polypeptide SEQ ID NO: 145 OsPolynucleotide transcript SEQ ID NO: 146 Pt Polypeptide SEQ ID NO: 147Pt Polynucleotide transcript SEQ ID NO: 148 Sb Polypeptide SEQ ID NO:149 Sb Polynucleotide transcript SEQ ID NO: 150 SCW08 At Polypeptide SEQID NO: 151 At Polynucleotide transcript SEQ ID NO: 152 Os PolypeptideSEQ ID NO: 153 Os Polynucleotide transcript SEQ ID NO: 154 PtPolypeptide SEQ ID NO: 155 Pt Polynucleotide transcript SEQ ID NO: 156SCW09 At Polypeptide SEQ ID NO: 157 At Polynucleotide transcript SEQ IDNO: 158 Mt Polypeptide SEQ ID NO: 159 Mt Polynucleotide transcript SEQID NO: 160 Os Polypeptide SEQ ID NO: 161 Os Polynucleotide transcriptSEQ ID NO: 162 Pt1Polypeptide SEQ ID NO: 163 Pt1 Polynucleotidetranscript SEQ ID NO: 164 Pt2 Polypeptide SEQ ID NO: 165 Pt2Polynucleotide transcript SEQ ID NO: 166 SCW10 At Polypeptide SEQ ID NO:167 At Polynucleotide transcript SEQ ID NO: 168 Mt Polypeptide SEQ IDNO: 169 Mt Polynucleotide transcript SEQ ID NO: 170 Os Polypeptide SEQID NO: 171 Os Polynucleotide transcript SEQ ID NO: 172 Pt1 PolypeptideSEQ ID NO: 173 Pt1 Polynucleotide transcript SEQ ID NO: 174 Pt2Polypeptide SEQ ID NO: 175 Pt2 Polynucleotide transcript SEQ ID NO: 176SCW11a Af Polypeptide SEQ ID NO: 177 At Polynucleotide transcript SEQ IDNO: 178 Mt Polypeptide SEQ ID NO: 179 Mt Polynucleotide transcript SEQID NO: 180 Os Polypeptide SEQ ID NO: 181 Os Polynucleotide transcriptSEQ ID NO: 182 Pt Polypeptide SEQ ID NO: 183 Pt Polynucleotidetranscript SEQ ID NO: 184 Sb Polynucleotide transcript SEQ ID NO: 411SCW11b Mt Polypeptide SEQ ID NO: 185 Mt Polynucleotide transcript SEQ IDNO: 186 Os Polypeptide SEQ ID NO: 187 Os Polynucleotide transcript SEQID NO: 188 Pt Polypeptide SEQ ID NO: 189 Pt Polynucleotide transcriptSEQ ID NO: 190 SCW13 At Polypeptide SEQ ID NO: 191 At Polynucleotidetranscript SEQ ID NO: 192 Mt Polypeptide SEQ ID NO: 193 MtPolynucleotide transcript SEQ ID NO: 194 Os Polypeptide SEQ ID NO: 195Os Polynucleotide transcript SEQ ID NO: 196 Pt Polypeptide SEQ ID NO:197 Pt Polynucleotide transcript SEQ ID NO: 198 Sb1 Polynucleotidetranscript SEQ ID NO: 412 Sb2 Polynucleotide transcript SEQ ID NO: 413SCW16 Os Polypeptide SEQ ID NO: 199 Os Polynucleotide transcript SEQ IDNO: 200 Sb Polynucleotide transcript SEQ ID NO: 414 SCW17 Os PolypeptideSEQ ID NO: 201 Os Polynucleotide transcript SEQ ID NO: 202 SCW21 AtPolypeptide SEQ ID NO: 203 At Polynucleotide transcript SEQ ID NO: 204Os Polypeptide SEQ ID NO: 205 Os Polynucleotide transcript SEQ ID NO:206 SCW22 At Polypeptide SEQ ID NO: 207 At Polynucleotide transcript SEQID NO: 208 Mt Polypeptide SEQ ID NO: 209 Mt Polynucleotide transcriptSEQ ID NO: 210 Os Polypeptide SEQ ID NO: 211 Os Polynucleotidetranscript SEQ ID NO: 212 Pt Polypeptide SEQ ID NO: 213 PtPolynucleotide transcript SEQ ID NO: 214 Sb1 Polypeptide SEQ ID NO: 215Sb1 Polynucleotide transcript SEQ ID NO: 216 Sb2 Polypeptide SEQ ID NO:217 Sb2 Polynucleotide transcript SEQ ID NO: 218 SCW23 At PolypeptideSEQ ID NO: 219 At Polynucleotide transcript SEQ ID NO: 220 OsPolypeptide SEQ ID NO: 221 Os Polynucleotide transcript SEQ ID NO: 222Pt Polypeptide SEQ ID NO: 223 Pt Polynucleotide transcript SEQ ID NO:224 Sb Polynucleotide transcript SEQ ID NO: 415 SCW26 At Polypeptide SEQID NO: 225 At Polynucleotide transcript SEQ ID NO: 226 Mt PolypeptideSEQ ID NO: 227 Mt Polynucleotide transcript SEQ ID NO: 228 OsPolypeptide SEQ ID NO: 229 Os Polynucleotide transcript SEQ ID NO: 230Pt1 Polypeptide SEQ ID NO: 231 Pt1 Polynucleotide transcript SEQ ID NO:232 Pt2 Polypeptide SEQ ID NO: 233 Pt2 Polynucleotide transcript SEQ IDNO: 234 Sb Polypeptide SEQ ID NO: 235 Sb Polynucleotide transcript SEQID NO: 236 SCW28 At Polypeptide SEQ ID NO: 237 At Polynucleotidetranscript SEQ ID NO: 238 Mt Polypeptide SEQ ID NO: 239 MtPolynucleotide transcript SEQ ID NO: 240 Os Polypeptide SEQ ID NO: 241Os Polynucleotide transcript SEQ ID NO: 242 Pt Polypeptide SEQ ID NO:243 Pt Polynucleotide transcript SEQ ID NO: 244 Sb Polypeptide SEQ IDNO: 245 Sb Polynucleotide transcript SEQ ID NO: 246 SCW32 At PolypeptideSEQ ID NO: 247 At Polynucleotide transcript SEQ ID NO: 248 MtPolypeptide SEQ ID NO: 249 Mt Polynucleotide transcript SEQ ID NO: 250Os Polypeptide SEQ ID NO: 251 Os Polynucleotide transcript SEQ ID NO:252 Pt Polypeptide SEQ ID NO: 253 Pt Polynucleotide transcript SEQ IDNO: 254 Sb Polynucleotide transcript SEQ ID NO: 416 SCW34 At PolypeptideSEQ ID NO: 255 At Polynucleotide transcript SEQ ID NO: 256 OsPolypeptide SEQ ID NO: 257 Os Polynucleotide transcript SEQ ID NO: 258Pt Polypeptide SEQ ID NO: 259 Pt Polynucleotide transcript SEQ ID NO:260 Sb Polypeptide SEQ ID NO: 261 Sb Polynucleotide transcript SEQ IDNO: 262 SCW38 At Polypeptide SEQ ID NO: 263 At Polynucleotide transcriptSEQ ID NO: 264 Mt Polypeptide SEQ ID NO: 265 Mt Polynucleotidetranscript SEQ ID NO: 266 Os Polypeptide SEQ ID NO: 267 OsPolynucleotide transcript SEQ ID NO: 268 Pt1 Polypeptide SEQ ID NO: 269Pt1 Polynucleotide transcript SEQ ID NO: 270 Pt2 Polypeptide SEQ ID NO:271 Pt2 Polynucleotide transcript SEQ ID NO: 272 Sb Polynucleotidetranscript SEQ ID NO: 417 SCW39 At Polypeptide SEQ ID NO: 273 AtPolynucleotide transcript SEQ ID NO: 274 Mt Polypeptide SEQ ID NO: 275Mt Polynucleotide transcript SEQ ID NO: 276 Os1 Polypeptide SEQ ID NO:277 Os1 Polynucleotide transcript SEQ ID NO: 278 Os2 Polypeptide SEQ IDNO: 279 Os2 Polynucleotide transcript SEQ ID NO: 280 Pt Polypeptide SEQID NO: 281 Pt Polynucleotide transcript SEQ ID NO: 282 Sb PolypeptideSEQ ID NO: 283 Sb Polynucleotide transcript SEQ ID NO: 284 SCW40 AtPolypeptide SEQ ID NO: 285 At Polynucleotide transcript SEQ ID NO: 286Mt Polypeptide SEQ ID NO: 287 Mt Polynucleotide transcript SEQ ID NO:288 Os Polypeptide SEQ ID NO: 289 Os Polynucleotide transcript SEQ IDNO: 290 Sb Polynucleotide transcript SEQ ID NO: 418 SCW41 At PolypeptideSEQ ID NO: 291 At Polynucleotide transcript SEQ ID NO: 292 MtPolypeptide SEQ ID NO: 293 Mt Polynucleotide transcript SEQ ID NO: 294Os Polypeptide SEQ ID NO: 295 Os Polynucleotide transcript SEQ ID NO:296 Pt Polypeptide SEQ ID NO: 297 Pt Polynucleotide transcript SEQ IDNO: 298 Sb Polypeptide SEQ ID NO: 299 Sb Polynucleotide transcript SEQID NO: 300 SCW43 At Polypeptide SEQ ID NO: 301 At Polynucleotidetranscript SEQ ID NO: 302 Os Polypeptide SEQ ID NO: 303 OsPolynucleotide transcript SEQ ID NO: 304 Sb Polypeptide SEQ ID NO: 305Sb Polynucleotide transcript SEQ ID NO: 306 SCW44 Mt1 Polypeptide SEQ IDNO: 307 Mt1 Polynucleotide transcript SEQ ID NO: 308 Mt2 Polypeptide SEQID NO: 309 Mt2 Polynucleotide transcript SEQ ID NO: 310 Os PolypeptideSEQ ID NO: 311 Os Polynucleotide transcript SEQ ID NO: 312 SbPolynucleotide transcript SEQ ID NO: 419 SCW45 At Polypeptide SEQ ID NO:313 At Polynucleotide transcript SEQ ID NO: 314 Mt Polypeptide SEQ IDNO: 315 Mt Polynucleotide transcript SEQ ID NO: 316 Os1 Polypeptide SEQID NO: 317 Os1 Polynucleotide transcript SEQ ID NO: 318 Os2 PolypeptideSEQ ID NO: 319 Os2 Polynucleotide transcript SEQ ID NO: 320 PtPolypeptide SEQ ID NO: 321 Pt Polynucleotide transcript SEQ ID NO: 322Sb Polynucleotide transcript SEQ ID NO: 420 SCW46 At Polypeptide SEQ IDNO: 323 At Polynucleotide transcript SEQ ID NO: 324 Os Polypeptide SEQID NO: 325 Os Polynucleotide transcript SEQ ID NO: 326 Pt1 PolypeptideSEQ ID NO: 327 Pt1 Polynucleotide transcript SEQ ID NO: 328 Pt2Polypeptide SEQ ID NO: 329 Pt2 Polynucleotide transcript SEQ ID NO: 330Sb Polypeptide SEQ ID NO: 331 Sb Polynucleotide transcript SEQ ID NO:332 SCW47 At Polypeptide SEQ ID NO: 333 At Polynucleotide transcript SEQID NO: 334 Mt Polypeptide SEQ ID NO: 335 Mt Polynucleotide transcriptSEQ ID NO: 336 Os Polypeptide SEQ ID NO: 337 Os Polynucleotidetranscript SEQ ID NO: 338 Pt Polypeptide SEQ ID NO: 339 PtPolynucleotide transcript SEQ ID NO: 340 Sb Polynucleotide transcriptSEQ ID NO: 421 SCW48 At Polypeptide SEQ ID NO: 341 At Polynucleotidetranscript SEQ ID NO: 342 Mt Polypeptide SEQ ID NO: 343 MtPolynucleotide transcript SEQ ID NO: 344 Os Polypeptide SEQ ID NO: 345Os Polynucleotide transcript SEQ ID NO: 346 Pt Polypeptide SEQ ID NO:347 Pt Polynucleotide transcript SEQ ID NO: 348 SCW49 At Polypeptide SEQID NO: 349 At Polynucleotide transcript SEQ ID NO: 350 Os PolypeptideSEQ ID NO: 351 Os Polynucleotide transcript SEQ ID NO: 352 SCW50 AtPolypeptide SEQ ID NO: 353 At Polynucleotide transcript SEQ ID NO: 354Mt Polypeptide SEQ ID NO: 355 Mt Polynucleotide transcript SEQ ID NO:356 Os Polypeptide SEQ ID NO: 357 Os Polynucleotide transcript SEQ IDNO: 358 Pt1 Polypeptide SEQ ID NO: 359 Pt1 Polynucleotide transcript SEQID NO: 360 Pt2 Polypeptide SEQ ID NO: 361 Pt2 Polynucleotide transcriptSEQ ID NO: 362 Sb Polynucleotide transcript SEQ ID NO: 422 SCW51 AtPolypeptide SEQ ID NO: 363 At Polynucleotide transcript SEQ ID NO: 364Mt Polypeptide SEQ ID NO: 365 Mt Polynucleotide transcript SEQ ID NO:366 Os Polypeptide SEQ ID NO: 367 Os Polynucleotide transcript SEQ IDNO: 368 Pt Polypeptide SEQ ID NO: 369 Pt Polynucleotide transcript SEQID NO: 370 SCW53 At1 Polypeptide SEQ ID NO: 371 At1 Polynucleotidetranscript SEQ ID NO: 372 At2 Polypeptide SEQ ID NO: 373 At2Polynucleotide transcript SEQ ID NO: 374 Mt Polypeptide SEQ ID NO: 375Mt Polynucleotide transcript SEQ ID NO: 376 Os1 Polypeptide SEQ ID NO:377 Os1 Polynucleotide transcript SEQ ID NO: 378 Os2 Polypeptide SEQ IDNO: 379 Os2 Polynucleotide transcript SEQ ID NO: 380 Pt Polypeptide SEQID NO: 381 Pt Polynucleotide transcript SEQ ID NO: 382 Sb Polynucleotidetranscript SEQ ID NO: 423 SCW54 At Polypeptide SEQ ID NO: 383 AtPolynucleotide transcript SEQ ID NO: 384 Os Polypeptide SEQ ID NO: 385Os Polynucleotide transcript SEQ ID NO: 386 SCW55 At Polypeptide SEQ IDNO: 387 At Polynucleotide transcript SEQ ID NO: 388 Os1 Polypeptide SEQID NO: 389 Os1 Polynucleotide transcript SEQ ID NO: 390 Os2 PolypeptideSEQ ID NO: 391 Os2 Polynucleotide transcript SEQ ID NO: 392 PtPolypeptide SEQ ID NO: 393 Pt Polynucleotide transcript SEQ ID NO: 394Sb Polypeptide SEQ ID NO: 395 Sb Polynucleotide transcript SEQ ID NO:396 SCW56 Os Polypeptide SEQ ID NO: 397 Os Polynucleotide transcript SEQID NO: 398 Sb Polypeptide SEQ ID NO: 399 Sb Polynucleotide transcriptSEQ ID NO: 400 SCW58 At1 Polypeptide SEQ ID NO: 401 At1 Polynucleotidetranscript SEQ ID NO: 402 At2 Polypeptide SEQ ID NO: 403 At2Polynucleotide transcript SEQ ID NO: 404 Os Polypeptide SEQ ID NO: 405Os Polynucleotide transcript SEQ ID NO: 406 Pt1 Polypeptide SEQ ID NO:407 Pt1 Polynucleotide transcript SEQ ID NO: 408 Pt2 Polypeptide SEQ IDNO: 409 Pt2 Polynucleotide transcript SEQ ID NO: 410 Sb Polynucleotidetranscript SEQ ID NO: 424

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) standard recombinant methods, (b) synthetic techniques, orcombinations thereof. In some embodiments, the polynucleotides of thepresent invention will be cloned, amplified, or otherwise constructedfrom a fungus or bacteria.

The nucleic acids may conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites may beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences may be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the polynucleotide sequence—isoptionally a vector, adapter, or linker for cloning and/or expression ofa polynucleotide of the present invention. Additional sequences may beadded to such cloning and/or expression sequences to optimize theirfunction in cloning and/or expression, to aid in isolation of thepolynucleotide, or to improve the introduction of the polynucleotideinto a cell. Typically, the length of a nucleic acid of the presentinvention less the length of its polynucleotide of the present inventionis less than 20 kilobase pairs, often less than 15 kb, and frequentlyless than 10 kb. Use of cloning vectors, expression vectors, adapters,and linkers is well known in the art. Exemplary nucleic acids includesuch vectors as: M13, lambda ZAP Express, lambda ZAP II, lambda gt10,lambda gt11, pBK-CMV, pBK-RSV, pBluescript II, lambda DASH II, lambdaEMBL 3, lambda EMBL 4, pWE15, SuperCos 1, SurfZap, Uni-ZAP, pBC, pBS+/−,pSG5, pBK, pCR-Script, pET, pSPUTK, p3'SS, pGEM, pSK+/−, pGEX, pSPORTIand II, pOPRSVI CAT, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo,pOG44, pOG45, pFRTβGAL, pNEOβGAL, pRS403, pRS404, pRS405, pRS406,pRS413, pRS414, pRS415, pRS416, lambda MOSSlox, and lambda MOSElox.Optional vectors for the present invention, include but are not limitedto, lambda ZAP II, and pGEX. For a description of various nucleic acidssee, e.g., Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (LaJolla, Calif.); and, Amersham Life Sciences, Inc, Catalog '97 (ArlingtonHeights, Ill.).

Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by methods such as the phosphotriestermethod of Narang, et al., (1979) Meth. Enzymol. 68:90-9; thephosphodiester method of Brown, et al., (1979) Meth. Enzymol. 68:109-51;the diethylphosphoramidite method of Beaucage, et al., (1981) Tetra.Letts. 22(20):1859-62; the solid phase phosphoramidite triester methoddescribed by Beaucage, et al., supra, e.g., using an automatedsynthesizer, e.g., as described in Needham-VanDevanter, et al., (1984)Nucleic Acids Res. 12:6159-68; and, the solid support method of U.S.Pat. No. 4,458,066. Chemical synthesis generally produces a singlestranded oligonucleotide. This may be converted into double stranded DNAby hybridization with a complementary sequence or by polymerization witha DNA polymerase using the single strand as a template. One of skillwill recognize that while chemical synthesis of DNA is limited tosequences of about 100 bases, longer sequences may be obtained by theligation of shorter sequences.

UTRs and Codon Preference

In general, translational efficiency has been found to be regulated byspecific sequence elements in the 5′ non-coding or untranslated region(5′ UTR) of the RNA. Positive sequence motifs include translationalinitiation consensus sequences (Kozak, (1987) Nucleic Acids Res.15:8125) and the 5<G>7 methyl GpppG RNA cap structure (Drummond, et al.,(1985) Nucleic Acids Res. 13:7375). Negative elements include stableintramolecular 5′ UTR stem-loop structures (Muesing, et al., (1987) Cell48:691) and AUG sequences or short open reading frames preceded by anappropriate AUG in the 5′ UTR (Kozak, supra, Rao, et al., (1988) Mol.and Cell. Biol. 8:284). Accordingly, the present invention provides 5′and/or 3′ UTR regions for modulation of translation of heterologouscoding sequences.

Further, the polypeptide-encoding segments of the polynucleotides of thepresent invention can be modified to alter codon usage. Altered codonusage can be employed to alter translational efficiency and/or tooptimize the coding sequence for expression in a desired host or tooptimize the codon usage in a heterologous sequence for expression inmaize. Codon usage in the coding regions of the polynucleotides of thepresent invention can be analyzed statistically using commerciallyavailable software packages such as “Codon Preference” available fromthe University of Wisconsin Genetics Computer Group. See, Devereaux, etal., (1984) Nucleic Acids Res. 12:387-395; or MacVector 4.1 (EastmanKodak Co., New Haven, Conn.). Thus, the present invention provides acodon usage frequency characteristic of the coding region of at leastone of the polynucleotides of the present invention. The number ofpolynucleotides (3 nucleotides per amino acid) that can be used todetermine a codon usage frequency can be any integer from 3 to thenumber of polynucleotides of the present invention as provided herein.Optionally, the polynucleotides will be full-length sequences. Anexemplary number of sequences for statistical analysis can be at least1, 5, 10, 20, 50 or 100.

Sequence Shuffling

The present invention provides methods for sequence shuffling usingpolynucleotides of the present invention, and compositions resultingtherefrom. Sequence shuffling is described in PCT publication number96/19256. See also, Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA94:4504-9; and Zhao, et al., (1998) Nature Biotech 16:258-61. Generally,sequence shuffling provides a means for generating libraries ofpolynucleotides having a desired characteristic, which can be selectedor screened for. Libraries of recombinant polynucleotides are generatedfrom a population of related sequence polynucleotides, which comprisesequence regions, which have substantial sequence identity and can behomologously recombined in vitro or in vivo. The population ofsequence-recombined polynucleotides comprises a subpopulation ofpolynucleotides which possess desired or advantageous characteristicsand which can be selected by a suitable selection or screening method.The characteristics can be any property or attribute capable of beingselected for or detected in a screening system, and may includeproperties of: an encoded protein, a transcriptional element, a sequencecontrolling transcription, RNA processing, RNA stability, chromatinconformation, translation, or other expression property of a gene ortransgene, a replicative element, a protein-binding element, or thelike, such as any feature which confers a selectable or detectableproperty. In some embodiments, the selected characteristic will be analtered K_(m) and/or K_(cat) over the wild-type protein as providedherein. In other embodiments, a protein or polynucleotide generated fromsequence shuffling will have a ligand binding affinity greater than thenon-shuffled wild-type polynucleotide. In yet other embodiments, aprotein or polynucleotide generated from sequence shuffling will have analtered pH optimum as compared to the non-shuffled wild-typepolynucleotide. The increase in such properties can be at least 110%,120%, 130%, 140% or greater than 150% of the wild-type value.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence coding for the desired polynucleotide of the present invention,for example a cDNA or a genomic sequence encoding a polypeptide longenough to code for an active protein of the present invention, can beused to construct a recombinant expression cassette which can beintroduced into the desired host cell. A recombinant expression cassettewill typically comprise a polynucleotide of the present inventionoperably linked to transcriptional initiation regulatory sequences whichwill direct the transcription of the polynucleotide in the intended hostcell, such as tissues of a transformed plant.

For example, plant expression vectors may include (1) a cloned plantgene under the transcriptional control of 5′ and 3′ regulatory sequencesand (2) a dominant selectable marker. Such plant expression vectors mayalso contain, if desired, a promoter regulatory region (e.g., oneconferring inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal.

A plant promoter fragment can be employed which will direct expressionof a polynucleotide of the present invention in all tissues of aregenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the 1′- or 2′-promoter derived fromT-DNA of Agrobacterium tumefaciens, the Smas promoter, the cinnamylalcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nospromoter, the rubisco promoter, the GRP1-8 promoter, the 35S promoterfrom cauliflower mosaic virus (CaMV), as described in Odell, et al.,(1985) Nature 313:810-2; rice actin (McElroy, et al., (1990) Plant Cell163-171); ubiquitin (Christensen, et al., (1992) Plant Mol. Biol.12:619-632, and Christensen, et al., (1992) Plant Mol. Biol. 18:675-89);pEMU (Last, et al., (1991) Theor. Appl. Genet. 81:581-8); MAS (Velten,et al., (1984) EMBO J. 3:2723-30); and maize H3 histone (Lepetit, etal., (1992) Mol. Gen. Genet. 231:276-85; and Atanassvoa, et al., (1992)Plant Journal 2(3):291-300); ALS promoter, as described in PCTApplication No. WO 96/30530; GOS2 (U.S. Pat. No. 6,504,083) and othertranscription initiation regions from various plant genes known to thoseof skill. For the present invention ubiquitin is the preferred promoterfor expression in monocot plants.

Alternatively, the plant promoter can direct expression of apolynucleotide of the present invention in a specific tissue or may beotherwise under more precise environmental or developmental control.Such promoters are referred to here as “inducible” promoters (Rab17,RAD29). Environmental conditions that may effect transcription byinducible promoters include pathogen attack, anaerobic conditions, orthe presence of light. Examples of inducible promoters are the Adh1promoter, which is inducible by hypoxia or cold stress, the Hsp70promoter, which is inducible by heat stress, and the PPDK promoter,which is inducible by light.

Examples of promoters under developmental control include promoters thatinitiate transcription only, or preferentially, in certain tissues, suchas leaves, roots, fruit, seeds, or flowers. The operation of a promotermay also vary depending on its location in the genome. Thus, aninducible promoter may become fully or partially constitutive in certainlocations.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from a varietyof plant genes, or from T-DNA. The 3′ end sequence to be added can bederived from, for example, the nopaline synthase or octopine synthasegenes, or alternatively from another plant gene, or less preferably fromany other eukaryotic gene. Examples of such regulatory elements include,but are not limited to, 3′ termination and/or polyadenylation regionssuch as those of the Agrobacterium tumefaciens nopaline synthase (nos)gene (Bevan, et al., (1983) Nucleic Acids Res. 12:369-85); the potatoproteinase inhibitor II (PINII) gene (Keil, et al., (1986) Nucleic AcidsRes. 14:5641-50; and An, et al., (1989) Plant Cell 1:115-22); and theCaMV 19S gene (Mogen, et al., (1990) Plant Cell 2:1261-72).

An intron sequence can be added to the 5′ untranslated region or thecoding sequence of the partial coding sequence to increase the amount ofthe mature message that accumulates in the cytosol. Inclusion of aspliceable intron in the transcription unit in both plant and animalexpression constructs has been shown to increase gene expression at boththe mRNA and protein levels up to 1000-fold (Buchman and Berg, (1988)Mol. Cell. Biol. 8:4395-4405; Callis, et al., (1987) Genes Dev.1:1183-200). Such intron enhancement of gene expression is typicallygreatest when placed near the 5′ end of the transcription unit. Use ofmaize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are knownin the art. See generally, THE MAIZE HANDBOOK, Chapter 116, Freeling andWalbot, eds., Springer, N.Y. (1994).

Plant signal sequences, including, but not limited to, signal-peptideencoding DNA/RNA sequences which target proteins to the extracellularmatrix of the plant cell (Dratewka-Kos, et al., (1989) J. Biol. Chem.264:4896-900), such as the Nicotiana plumbaginifolia extension gene(DeLoose, et al., (1991) Gene 99:95-100); signal peptides which targetproteins to the vacuole, such as the sweet potato sporamin gene(Matsuka, et al., (1991) Proc. Natl. Acad. Sci. USA 88:834) and thebarley lectin gene (Wilkins, et al., (1990) Plant Cell, 2:301-13);signal peptides which cause proteins to be secreted, such as that ofPRIb (Lind, et al., (1992) Plant Mol. Biol. 18:47-53) or the barleyalpha amylase (BAA) (Rahmatullah, et al., (1989) Plant Mol. Biol.12:119, and hereby incorporated by reference), or signal peptides whichtarget proteins to the plastids such as that of rapeseed enoyl-Acpreductase (Verwaert, et al., (1994) Plant Mol. Biol. 26:189-202) areuseful in the invention. The barley alpha amylase signal sequence fusedto the SCW polynucleotide is the preferred construct for expression inmaize for the present invention.

The vector comprising the sequences from a polynucleotide of the presentinvention will typically comprise a marker gene, which confers aselectable phenotype on plant cells. Usually, the selectable marker genewill encode antibiotic resistance, with suitable genes including genescoding for resistance to the antibiotic spectinomycin (e.g., the aadagene), the streptomycin phosphotransferase (SPT) gene coding forstreptomycin resistance, the neomycin phosphotransferase (NPTII) geneencoding kanamycin or geneticin resistance, the hygromycinphosphotransferase (HPT) gene coding for hygromycin resistance, genescoding for resistance to herbicides which act to inhibit the action ofacetolactate synthase (ALS), in particular the sulfonylurea-typeherbicides (e.g., the acetolactate synthase (ALS) gene containingmutations leading to such resistance in particular the S4 and/or Hramutations), genes coding for resistance to herbicides which act toinhibit action of glutamine synthase, such as phosphinothricin or basta(e.g., the bar gene), or other such genes known in the art. The bar geneencodes resistance to the herbicide basta, and the ALS gene encodesresistance to the herbicide chlorsulfuron.

Typical vectors useful for expression of genes in higher plants are wellknown in the art and include vectors derived from the tumor-inducing(Ti) plasmid of Agrobacterium tumefaciens described by Rogers, et al.,(1987) Meth. Enzymol. 153:253-77. These vectors are plant integratingvectors in that on transformation, the vectors integrate a portion ofvector DNA into the genome of the host plant. Exemplary A. tumefaciensvectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl, et al.,(1987) Gene 61:1-11, and Berger, et al., (1989) Proc. Natl. Acad. Sci.USA, 86:8402-6. Another useful vector herein is plasmid pBI101.2 that isavailable from CLONTECH Laboratories, Inc. (Palo Alto, Calif.).

Expression of Proteins in Host Cells

Using the nucleic acids of the present invention, one may express aprotein of the present invention in a recombinantly engineered cell suchas bacteria, yeast, insect, mammalian, or preferably plant cells. Thecells produce the protein in a non-natural condition (e.g., in quantity,composition, location, and/or time), because they have been geneticallyaltered through human intervention to do so.

It is expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of a nucleic acidencoding a protein of the present invention. No attempt to describe indetail the various methods known for the expression of proteins inprokaryotes or eukaryotes will be made.

In brief summary, the expression of isolated nucleic acids encoding aprotein of the present invention will typically be achieved by operablylinking, for example, the DNA or cDNA to a promoter (which is eitherconstitutive or inducible), followed by incorporation into an expressionvector. The vectors can be suitable for replication and integration ineither prokaryotes or eukaryotes. Typical expression vectors containtranscription and translation terminators, initiation sequences, andpromoters useful for regulation of the expression of the DNA encoding aprotein of the present invention. To obtain high level expression of acloned gene, it is desirable to construct expression vectors whichcontain, at the minimum, a strong promoter, such as ubiquitin, to directtranscription, a ribosome binding site for translational initiation, anda transcription/translation terminator. Constitutive promoters areclassified as providing for a range of constitutive expression. Thus,some are weak constitutive promoters, and others are strong constitutivepromoters. Generally, by “weak promoter” is intended a promoter thatdrives expression of a coding sequence at a low level. By “low level” isintended at levels of about 1/10,000 transcripts to about 1/100,000transcripts to about 1/500,000 transcripts. Conversely, a “strongpromoter” drives expression of a coding sequence at a “high level,” orabout 1/10 transcripts to about 1/100 transcripts to about 1/1,000transcripts.

One of skill would recognize that modifications could be made to aprotein of the present invention without diminishing its biologicalactivity. Some modifications may be made to facilitate the cloning,expression, or incorporation of the targeting molecule into a fusionprotein. Such modifications are well known to those of skill in the artand include, for example, a methionine added at the amino terminus toprovide an initiation site, or additional amino acids (e.g., poly His)placed on either terminus to create conveniently located restrictionsites or termination codons or purification sequences.

Expression in Prokaryotes

Prokaryotic cells may be used as hosts for expression. Prokaryotes mostfrequently are represented by various strains of E. coli; however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences, include such commonly used promoters asthe beta lactamase (penicillinase) and lactose (lac) promoter systems(Chang, et al., (1977) Nature 198:1056), the tryptophan (trp) promotersystem (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057) and thelambda derived P L promoter and N-gene ribosome binding site (Shimatake,et al., (1981) Nature 292:128). The inclusion of selection markers inDNA vectors transfected in E. coli is also useful. Examples of suchmarkers include genes specifying resistance to ampicillin, tetracycline,or chloramphenicol.

The vector is selected to allow introduction of the gene of interestinto the appropriate host cell. Bacterial vectors are typically ofplasmid or phage origin. Appropriate bacterial cells are infected withphage vector particles or transfected with naked phage vector DNA. If aplasmid vector is used, the bacterial cells are transfected with theplasmid vector DNA. Expression systems for expressing a protein of thepresent invention are available using Bacillus sp. and Salmonella(Palva, et al., (1983) Gene 22:229-35; Mosbach, et al., (1983) Nature302:543-5). The pGEX-4T-1 plasmid vector from Pharmacia is the preferredE. coli expression vector for the present invention.

Expression in Eukaryotes

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained briefly below, the present invention can be expressedin these eukaryotic systems. In some embodiments,transformed/transfected plant cells, as discussed infra, are employed asexpression systems for production of the proteins of the instantinvention.

Synthesis of heterologous proteins in yeast is well known. Sherman, etal., (1982) METHODS IN YEAST GENETICS, Cold Spring Harbor Laboratory isa well recognized work describing the various methods available toproduce the protein in yeast. Two widely utilized yeasts for productionof eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.Vectors, strains, and protocols for expression in Saccharomyces andPichia are known in the art and available from commercial suppliers(e.g., Invitrogen). Suitable vectors usually have expression controlsequences, such as promoters, including 3-phosphoglycerate kinase oralcohol oxidase, and an origin of replication, termination sequences andthe like as desired.

A protein of the present invention, once expressed, can be isolated fromyeast by lysing the cells and applying standard protein isolationtechniques to the lysates or the pellets. The monitoring of thepurification process can be accomplished by using Western blottechniques or radioimmunoassay of other standard immunoassay techniques.

The sequences encoding proteins of the present invention can also beligated to various expression vectors for use in transfecting cellcultures of, for instance, mammalian, insect, or plant origin. Mammaliancell systems often will be in the form of monolayers of cells althoughmammalian cell suspensions may also be used. A number of suitable hostcell lines capable of expressing intact proteins have been developed inthe art, and include the HEK293, BHK21, and CHO cell lines. Expressionvectors for these cells can include expression control sequences, suchas an origin of replication, a promoter (e.g., the CMV promoter, a HSVtk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer(Queen, et al., (1986) Immunol. Rev. 89:49), and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites (e.g., an SV40 large T Ag poly A addition site),and transcriptional terminator sequences. Other animal cells useful forproduction of proteins of the present invention are available, forinstance, from the American Type Culture Collection Catalogue of CellLines and Hybridomas (7^(th) ed., 1992).

Appropriate vectors for expressing proteins of the present invention ininsect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, armyworm, moth, andDrosophila cell lines such as a Schneider cell line (see, e.g.,Schneider, (1987) J. Embryol. Exp. Morphol. 27:353-65).

As with yeast, when higher animal or plant host cells are employed,polyadenlyation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenlyation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (Sprague, et al.,(1983) J. Virol. 45:773-81). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors (Saveria-Campo,“Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector,” in DNACLONING: A PRACTICAL APPROACH, vol. II, Glover, ed., IRL Press,Arlington, Va., pp. 213-38 (1985)).

In addition, the gene for SCW placed in the appropriate plant expressionvector can be used to transform plant cells. The polypeptide can then beisolated from plant callus or the transformed cells can be used toregenerate transgenic plants. Such transgenic plants can be harvested,and the appropriate tissues (seed or leaves, for example) can besubjected to large scale protein extraction and purification techniques.

Plant Transformation Methods

Numerous methods for introducing foreign genes into plants are known andcan be used to insert a SCW polynucleotide into a plant host, includingbiological and physical plant transformation protocols. See, e.g., Miki,et al., “Procedure for Introducing Foreign DNA into Plants,” in METHODSIN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick and Thompson, eds.,CRC Press, Inc., Boca Raton, pp. 67-88 (1993). The methods chosen varywith the host plant, and include chemical transfection methods such ascalcium phosphate, microorganism-mediated gene transfer such asAgrobacterium (Horsch, et al., (1985) Science 227:1229-31),electroporation, micro-injection, and biolistic bombardment.

Expression cassettes and vectors and in vitro culture methods for plantcell or tissue transformation and regeneration of plants are known andavailable. See, e.g., Gruber, et al., “Vectors for PlantTransformation,” in METHODS IN PLANT MOLECULAR BIOLOGY ANDBIOTECHNOLOGY, supra, pp. 89-119.

The isolated polynucleotides or polypeptides may be introduced into theplant by one or more techniques typically used for direct delivery intocells. Such protocols may vary depending on the type of organism, cell,plant or plant cell, i.e., monocot or dicot, targeted for genemodification. Suitable methods of transforming plant cells includemicroinjection (Crossway, et al., (1986) Biotechniques 4:320-334; andU.S. Pat. No. 6,300,543), electroporation (Riggs, et al., (1986) Proc.Natl. Acad. Sci. USA 83:5602-5606, direct gene transfer (Paszkowski, etal., (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration(see, for example, Sanford, et al., U.S. Pat. No. 4,945,050; WO91/10725; and McCabe, et al., (1988) Biotechnology 6:923-926). Also see,Tomes, et al., Direct DNA Transfer into Intact Plant Cells ViaMicroprojectile Bombardment. pp. 197-213 in Plant Cell, Tissue and OrganCulture, Fundamental Methods eds. O. L. Gamborg & G. C. Phillips,Springer-Verlag Berlin Heidelberg N.Y., 1995; U.S. Pat. No. 5,736,369(meristem); Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477;Sanford, et al., (1987) Particulate Science and Technology 5:27-37(onion); Christou, et al., (1988) Plant Physiol. 87:671-674 (soybean);Datta, et al., (1990) Biotechnology 8:736-740 (rice); Klein, et al.,(1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein, et al.,(1988) Biotechnology 6:559-563 (maize); WO 91/10725 (maize); Klein, etal., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990)Biotechnology 8:833-839; and Gordon-Kamm, et al., (1990) Plant Cell2:603-618 (maize); Hooydaas-Van Slogteren & Hooykaas (1984) Nature(London) 311:763-764; Bytebier, et al., (1987) Proc. Natl. Acad. Sci.USA 84:5345-5349 (Liliaceae); De Wet, et al., (1985) In The ExperimentalManipulation of Ovule Tissues, ed. G. P. Chapman, et al., pp. 197-209;Longman, N.Y. (pollen); Kaeppler, et al., (1990) Plant Cell Reports9:415-418; and Kaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); U.S. Pat. No. 5,693,512 (sonication);D'Halluin, et al., (1992) Plant Cell 4:1495-1505 (electroporation); Li,et al., (1993) Plant Cell Reports 12:250-255; and Christou and Ford(1995) Annals of Botany 75:407-413 (rice); Osjoda, et al., (1996) NatureBiotech. 14:745-750; Agrobacterium mediated maize transformation (U.S.Pat. No. 5,981,840); silicon carbide whisker methods (Frame, et al.,(1994) Plant J. 6:941-948); laser methods (Guo, et al., (1995)Physiologia Plantarum 93:19-24); sonication methods (Bao, et al., (1997)Ultrasound in Medicine & Biology 23:953-959; Finer and Finer (2000) LettAppl Microbiol. 30:406-10; Amoah, et al., (2001) J Exp Bot 52:1135-42);polyethylene glycol methods (Krens, et al., (1982) Nature 296:72-77);protoplasts of monocot and dicot cells can be transformed usingelectroporation (Fromm, et al., (1985) Proc. Natl. Acad. Sci. USA82:5824-5828) and microinjection (Crossway, et al., (1986) Mol. Gen.Genet. 202:179-185); all of which are herein incorporated by reference.

Agrobacterium-Mediated Transformation

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria, which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of plants. See, e.g., Kado,(1991) Crit. Rev. Plant Sci. 10:1. Descriptions of the Agrobacteriumvector systems and methods for Agrobacterium-mediated gene transfer areprovided in Gruber, et al., supra; Miki, et al., supra; and Moloney, etal., (1989) Plant Cell Reports 8:238.

Similarly, the gene can be inserted into the T-DNA region of a Ti or Riplasmid derived from A. tumefaciens or A. rhizogenes, respectively.Thus, expression cassettes can be constructed as above, using theseplasmids. Many control sequences are known which when coupled to aheterologous coding sequence and transformed into a host organism showfidelity in gene expression with respect to tissue/organ specificity ofthe original coding sequence. See, e.g., Benfey and Chua, (1989) Science244:174-81. Particularly suitable control sequences for use in theseplasmids are promoters for constitutive leaf-specific expression of thegene in the various target plants. Other useful control sequencesinclude a promoter and terminator from the nopaline synthase gene (NOS).The NOS promoter and terminator are present in the plasmid pARC2,available from the American Type Culture Collection and designated ATCC67238. If such a system is used, the virulence (vir) gene from eitherthe Ti or Ri plasmid must also be present, either along with the T-DNAportion, or via a binary system where the vir gene is present on aseparate vector. Such systems, vectors for use therein, and methods oftransforming plant cells are described in U.S. Pat. No. 4,658,082; U.S.patent application Ser. No. 913,914, filed Oct. 1, 1986, as referencedin U.S. Pat. No. 5,262,306, issued Nov. 16, 1993; and Simpson, et al.,(1986) Plant Mol. Biol. 6:403-15 (also referenced in the '306 patent);all incorporated by reference in their entirety.

Once constructed, these plasmids can be placed into A. rhizogenes or A.tumefaciens and these vectors used to transform cells of plant species,which are ordinarily susceptible to Fusarium or Alternaria infection.Several other transgenic plants are also contemplated by the presentinvention including but not limited to soybean, corn, sorghum, alfalfa,rice, clover, cabbage, banana, coffee, celery, tobacco, cowpea, cotton,melon and pepper. The selection of either A. tumefaciens or A.rhizogenes will depend on the plant being transformed thereby. Ingeneral A. tumefaciens is the preferred organism for transformation.Most dicotyledonous plants, some gymnosperms, and a few monocotyledonousplants (e.g., certain members of the Liliales and Arales) aresusceptible to infection with A. tumefaciens. A. rhizogenes also has awide host range, embracing most dicots and some gymnosperms, whichincludes members of the Leguminosae, Compositae, and Chenopodiaceae.Monocot plants can now be transformed with some success. EP ApplicationNumber 604 662 A1 discloses a method for transforming monocots usingAgrobacterium. EP Application Number 672 752 A1 discloses a method fortransforming monocots with Agrobacterium using the scutellum of immatureembryos. Ishida, et al., discuss a method for transforming maize byexposing immature embryos to A. tumefaciens (Nature Biotechnology14:745-50 (1996)).

Once transformed, these cells can be used to regenerate transgenicplants. For example, whole plants can be infected with these vectors bywounding the plant and then introducing the vector into the wound site.Any part of the plant can be wounded, including leaves, stems and roots.Alternatively, plant tissue, in the form of an explant, such ascotyledonary tissue or leaf disks, can be inoculated with these vectors,and cultured under conditions, which promote plant regeneration. Rootsor shoots transformed by inoculation of plant tissue with A. rhizogenesor A. tumefaciens, containing the gene coding for the fumonisindegradation enzyme, can be used as a source of plant tissue toregenerate fumonisin-resistant transgenic plants, either via somaticembryogenesis or organogenesis. Examples of such methods forregenerating plant tissue are disclosed in Shahin, Theor. Appl. Genet.69:235-40 (1985); U.S. Pat. No. 4,658,082; Simpson, et al., supra; andU.S. patent application Ser. Nos. 913,913 and 913,914, both filed Oct.1, 1986, as referenced in U.S. Pat. No. 5,262,306, issued Nov. 16, 1993,the entire disclosures therein incorporated herein by reference.

Direct Gene Transfer

Despite the fact that the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice (Hiei, et al.,(1994) The Plant Journal 6:271-82). Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation, where DNA is carried on thesurface of microprojectiles measuring about 1 to 4 μm. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate the plant cell walls and membranes (Sanford, etal., (1987) Part. Sci. Technol. 5:27; Sanford, (1988) Trends Biotech6:299; Sanford, (1990) Physiol. Plant 79:206; and Klein, et al., (1992)Biotechnology 10:268).

Another method for physical delivery of DNA to plants is sonication oftarget cells as described in Zang, et al., (1991) BioTechnology 9:996.Alternatively, liposome or spheroplast fusions have been used tointroduce expression vectors into plants. See, e.g., Deshayes, et al.,(1985) EMBO J. 4:2731; and Christou, et al., (1987) Proc. Natl. Acad.Sci. USA 84:3962. Direct uptake of DNA into protoplasts using CaCl₂precipitation, polyvinyl alcohol, or poly-L-ornithine has also beenreported. See, e.g., Hain, et al., (1985) Mol. Gen. Genet. 199:161; andDraper, et al., (1982) Plant Cell Physiol. 23:451.

Electroporation of protoplasts and whole cells and tissues has also beendescribed. See, e.g., Donn, et al., (1990) in Abstracts of the VIIthInt'l. Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53;D'Halluin, et al., (1992) Plant Cell 4:1495-505; and Spencer, et al.,(1994) Plant Mol. Biol. 24:51-61.

Increasing the Activity and/or Level of a SCW Polypeptide

Methods are provided to increase the activity and/or level of the SCWpolypeptide of the invention. An increase in the level and/or activityof the SCW polypeptide of the invention can be achieved by providing tothe plant an SCW polypeptide. The SCW polypeptide can be provided byintroducing the amino acid sequence encoding the SCW polypeptide intothe plant, introducing into the plant a nucleotide sequence encoding anSCW polypeptide or alternatively by selecting for different variants ofthe genomic locus encoding the SCW polypeptide of the invention.

As discussed elsewhere herein, many methods are known the art forproviding a polypeptide to a plant including, but not limited to, directintroduction of the polypeptide into the plant, introducing into theplant (transiently or stably) a polynucleotide construct encoding apolypeptide having secondary cell wall development activity. It is alsorecognized that the methods of the invention may employ a polynucleotidethat is not capable of directing, in the transformed plant, theexpression of a protein or an RNA. Thus, the level and/or activity of anSCW polypeptide may be increased by altering the gene encoding the SCWpolypeptide or its promoter. See, e.g., Kmiec, U.S. Pat. No. 5,565,350;Zarling, et al., PCT/US93/03868. Therefore mutagenized plants that carrymutations in SCW genes, where the mutations increase expression of theSCW gene or increase the plant growth and/or secondary cell walldevelopment activity of the encoded SCW polypeptide are provided.

Reducing the Activity and/or Level of a SCW Polypeptide

Methods are provided to reduce or eliminate the activity of an SCWpolypeptide of the invention by transforming a plant cell with anexpression cassette that expresses a polynucleotide that inhibits theexpression of the SCW polypeptide. The polynucleotide may inhibit theexpression of the SCW polypeptide directly, by preventing translation ofthe SCW messenger RNA, or indirectly, by encoding a polypeptide thatinhibits the transcription or translation of a SCW gene encoding a SCWpolypeptide. Methods for inhibiting or eliminating the expression of agene in a plant are well known in the art, and any such method may beused in the present invention to inhibit the expression of an SCWpolypeptide.

In accordance with the present invention, the expression of a SCWpolypeptide is inhibited if the protein level of the SCW polypeptide isless than 70% of the protein level of the same SCW polypeptide in aplant that has not been genetically modified or mutagenized to inhibitthe expression of that SCW polypeptide. In particular embodiments of theinvention, the protein level of the SCW polypeptide in a modified plantaccording to the invention is less than 60%, less than 50%, less than40%, less than 30%, less than 20%, less than 10%, less than 5% or lessthan 2% of the protein level of the same SCW polypeptide in a plant thatis not a mutant or that has not been genetically modified to inhibit theexpression of that SCW polypeptide. The expression level of the SCWpolypeptide may be measured directly, for example, by assaying for thelevel of SCW polypeptide expressed in the plant cell or plant, orindirectly, for example, by measuring the plant growth and/or secondarycell wall development activity of the SCW polypeptide in the plant cellor plant, or by measuring the biomass in the plant. Methods forperforming such assays are described elsewhere herein.

In other embodiments of the invention, the activity of the SCWpolypeptides is reduced or eliminated by transforming a plant cell withan expression cassette comprising a polynucleotide encoding apolypeptide that inhibits the activity of a SCW polypeptide. The plantgrowth and/or secondary cell wall development activity of a SCWpolypeptide is inhibited according to the present invention if the plantgrowth and/or secondary cell wall development activity of the SCWpolypeptide is less than 70% of the plant growth and/or secondary cellwall development activity of the same SCW polypeptide in a plant thathas not been modified to inhibit the plant growth and/or secondary cellwall development activity of that SCW polypeptide. In particularembodiments of the invention, the plant growth and/or secondary cellwall development activity of the SCW polypeptide in a modified plantaccording to the invention is less than 60%, less than 50%, less than40%, less than 30%, less than 20%, less than 10%, or less than 5% of theplant growth and/or secondary cell wall development activity of the sameSCW polypeptide in a plant that that has not been modified to inhibitthe expression of that SCW polypeptide. The plant growth and/orsecondary cell wall development activity of an SCW polypeptide is“eliminated” according to the invention when it is not detectable by theassay methods described elsewhere herein. Methods of determining theplant growth and/or secondary cell wall development activity of an SCWpolypeptide are described elsewhere herein.

In other embodiments, the activity of an SCW polypeptide may be reducedor eliminated by disrupting the gene encoding the SCW polypeptide. Theinvention encompasses mutagenized plants that carry mutations in SCWgenes, where the mutations reduce expression of the SCW gene or inhibitthe plant growth and/or secondary cell wall development activity of theencoded SCW polypeptide.

Thus, many methods may be used to reduce or eliminate the activity of anSCW polypeptide. In addition, more than one method may be used to reducethe activity of a single SCW polypeptide. Non-limiting examples ofmethods of reducing or eliminating the expression of SCW polypeptidesare given below.

1. Polynucleotide-Based Methods:

In some embodiments of the present invention, a plant is transformedwith an expression cassette that is capable of expressing apolynucleotide that inhibits the expression of an SCW polypeptide of theinvention. The term “expression” as used herein refers to thebiosynthesis of a gene product, including the transcription and/ortranslation of said gene product. For example, for the purposes of thepresent invention, an expression cassette capable of expressing apolynucleotide that inhibits the expression of at least one SCWpolypeptide is an expression cassette capable of producing an RNAmolecule that inhibits the transcription and/or translation of at leastone SCW polypeptide of the invention. The “expression” or “production”of a protein or polypeptide from a DNA molecule refers to thetranscription and translation of the coding sequence to produce theprotein or polypeptide, while the “expression” or “production” of aprotein or polypeptide from an RNA molecule refers to the translation ofthe RNA coding sequence to produce the protein or polypeptide.

Examples of polynucleotides that inhibit the expression of an SCWpolypeptide are given below.

i. Sense Suppression/Cosuppression

In some embodiments of the invention, inhibition of the expression of aSCW polypeptide may be obtained by sense suppression or cosuppression.For cosuppression, an expression cassette is designed to express an RNAmolecule corresponding to all or part of a messenger RNA encoding an SCWpolypeptide in the “sense” orientation. Over expression of the RNAmolecule can result in reduced expression of the native gene.Accordingly, multiple plant lines transformed with the cosuppressionexpression cassette are screened to identify those that show thegreatest inhibition of SCW polypeptide expression.

The polynucleotide used for cosuppression may correspond to all or partof the sequence encoding the SCW polypeptide, all or part of the 5′and/or 3′ untranslated region of an SCW polypeptide transcript, or allor part of both the coding sequence and the untranslated regions of atranscript encoding an SCW polypeptide. In some embodiments where thepolynucleotide comprises all or part of the coding region for the SCWpolypeptide, the expression cassette is designed to eliminate the startcodon of the polynucleotide so that no protein product will betranslated.

Cosuppression may be used to inhibit the expression of plant genes toproduce plants having undetectable protein levels for the proteinsencoded by these genes. See, for example, Broin, et al., (2002) PlantCell 14:1417-1432. Cosuppression may also be used to inhibit theexpression of multiple proteins in the same plant. See, for example,U.S. Pat. No. 5,942,657. Methods for using cosuppression to inhibit theexpression of endogenous genes in plants are described in Flavell, etal., (1994) Proc. Natl. Acad. Sci. USA 91:3490-3496; Jorgensen, et al.,(1996) Plant Mol. Biol. 31:957-973; Johansen and Carrington (2001) PlantPhysiol. 126:930-938; Broin, et al., (2002) Plant Cell 14:1417-1432;Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; Yu, et al.,(2003) Phytochemistry 63:753-763; and U.S. Pat. Nos. 5,034,323,5,283,184, and 5,942,657; each of which is herein incorporated byreference. The efficiency of cosuppression may be increased by includinga poly-dT region in the expression cassette at a position 3′ to thesense sequence and 5′ of the polyadenylation signal. See, US PatentApplication Publication Number 20020048814, herein incorporated byreference. Typically, such a nucleotide sequence has substantialsequence identity to the sequence of the transcript of the endogenousgene, optimally greater than about 65% sequence identity, more optimallygreater than about 85% sequence identity, most optimally greater thanabout 95% sequence identity. See, U.S. Pat. Nos. 5,283,184 and5,034,323; herein incorporated by reference.

ii. Antisense Suppression

In some embodiments of the invention, inhibition of the expression ofthe SCW polypeptide may be obtained by antisense suppression. Forantisense suppression, the expression cassette is designed to express anRNA molecule complementary to all or part of a messenger RNA encodingthe SCW polypeptide. Over expression of the antisense RNA molecule canresult in reduced expression of the native gene. Accordingly, multipleplant lines transformed with the antisense suppression expressioncassette are screened to identify those that show the greatestinhibition of SCW polypeptide expression.

The polynucleotide for use in antisense suppression may correspond toall or part of the complement of the sequence encoding the SCWpolypeptide, all or part of the complement of the 5′ and/or 3′untranslated region of the SCW transcript, or all or part of thecomplement of both the coding sequence and the untranslated regions of atranscript encoding the SCW polypeptide. In addition, the antisensepolynucleotide may be fully complementary (i.e., 100% identical to thecomplement of the target sequence) or partially complementary (i.e.,less than 100% identical to the complement of the target sequence) tothe target sequence. Antisense suppression may be used to inhibit theexpression of multiple proteins in the same plant. See, for example,U.S. Pat. No. 5,942,657. Furthermore, portions of the antisensenucleotides may be used to disrupt the expression of the target gene.Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200nucleotides, 300, 400, 450, 500, 550 or greater may be used. Methods forusing antisense suppression to inhibit the expression of endogenousgenes in plants are described, for example, in Liu, et al., (2002) PlantPhysiol. 129:1732-1743 and U.S. Pat. Nos. 5,759,829 and 5,942,657, eachof which is herein incorporated by reference. Efficiency of antisensesuppression may be increased by including a poly-dT region in theexpression cassette at a position 3′ to the antisense sequence and 5′ ofthe polyadenylation signal. See, US Patent Application PublicationNumber 20020048814, herein incorporated by reference.

iii. Double-Stranded RNA Interference

In some embodiments of the invention, inhibition of the expression of aSCW polypeptide may be obtained by double-stranded RNA (dsRNA)interference. For dsRNA interference, a sense RNA molecule like thatdescribed above for cosuppression and an antisense RNA molecule that isfully or partially complementary to the sense RNA molecule are expressedin the same cell, resulting in inhibition of the expression of thecorresponding endogenous messenger RNA.

Expression of the sense and antisense molecules can be accomplished bydesigning the expression cassette to comprise both a sense sequence andan antisense sequence. Alternatively, separate expression cassettes maybe used for the sense and antisense sequences. Multiple plant linestransformed with the dsRNA interference expression cassette orexpression cassettes are then screened to identify plant lines that showthe greatest inhibition of SCW polypeptide expression. Methods for usingdsRNA interference to inhibit the expression of endogenous plant genesare described in Waterhouse, et al., (1998) Proc. Natl. Acad. Sci. USA95:13959-13964, Liu, et al., (2002) Plant Physiol. 129:1732-1743, and WO99/49029, WO 99/53050, WO 99/61631, and WO 00/49035; each of which isherein incorporated by reference.

iv. Hairpin RNA Interference and Intron-Containing Hairpin RNAInterference

In some embodiments of the invention, inhibition of the expression ofone or a SCW polypeptide may be obtained by hairpin RNA (hpRNA)interference or intron-containing hairpin RNA (ihpRNA) interference.These methods are highly efficient at inhibiting the expression ofendogenous genes. See, Waterhouse and Helliwell (2003) Nat. Rev. Genet.4:29-38 and the references cited therein.

For hpRNA interference, the expression cassette is designed to expressan RNA molecule that hybridizes with itself to form a hairpin structurethat comprises a single-stranded loop region and a base-paired stem. Thebase-paired stem region comprises a sense sequence corresponding to allor part of the endogenous messenger RNA encoding the gene whoseexpression is to be inhibited, and an antisense sequence that is fullyor partially complementary to the sense sequence. Thus, the base-pairedstem region of the molecule generally determines the specificity of theRNA interference. hpRNA molecules are highly efficient at inhibiting theexpression of endogenous genes, and the RNA interference they induce isinherited by subsequent generations of plants. See, for example, Chuangand Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990;Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; andWaterhouse and Helliwell (2003) Nat. Rev. Genet. 4:29-38. Methods forusing hpRNA interference to inhibit or silence the expression of genesare described, for example, in Chuang and Meyerowitz (2000) Proc. Natl.Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Physiol.129:1723-1731; Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4:29-38;Pandolfini, et al., BMC Biotechnology 3:7, and US Patent ApplicationPublication Number 20030175965; each of which is herein incorporated byreference. A transient assay for the efficiency of hpRNA constructs tosilence gene expression in vivo has been described by Panstruga, et al.,(2003) Mol. Biol. Rep. 30:135-140, herein incorporated by reference.

For ihpRNA, the interfering molecules have the same general structure asfor hpRNA, but the RNA molecule additionally comprises an intron that iscapable of being spliced in the cell in which the ihpRNA is expressed.The use of an intron minimizes the size of the loop in the hairpin RNAmolecule following splicing, and this increases the efficiency ofinterference. See, for example, Smith, et al., (2000) Nature407:319-320. In fact, Smith, et al., show 100% suppression of endogenousgene expression using ihpRNA-mediated interference. Methods for usingihpRNA interference to inhibit the expression of endogenous plant genesare described, for example, in Smith, et al., (2000) Nature 407:319-320;Wesley, et al., (2001) Plant J. 27:581-590; Wang and Waterhouse (2001)Curr. Opin. Plant Biol. 5:146-150; Waterhouse and Helliwell (2003) Nat.Rev. Genet. 4:29-38; Helliwell and Waterhouse (2003) Methods 30:289-295,and US Patent Application Publication Number 20030180945, each of whichis herein incorporated by reference.

The expression cassette for hpRNA interference may also be designed suchthat the sense sequence and the antisense sequence do not correspond toan endogenous RNA. In this embodiment, the sense and antisense sequenceflank a loop sequence that comprises a nucleotide sequence correspondingto all or part of the endogenous messenger RNA of the target gene. Thus,it is the loop region that determines the specificity of the RNAinterference. See, for example, WO 02/00904, herein incorporated byreference.

v. Amplicon-Mediated Interference

Amplicon expression cassettes comprise a plant virus-derived sequencethat contains all or part of the target gene but generally not all ofthe genes of the native virus. The viral sequences present in thetranscription product of the expression cassette allow the transcriptionproduct to direct its own replication. The transcripts produced by theamplicon may be either sense or antisense relative to the targetsequence (i.e., the messenger RNA for the SCW polypeptide). Methods ofusing amplicons to inhibit the expression of endogenous plant genes aredescribed, for example, in Angell and Baulcombe (1997) EMBO J.16:3675-3684, Angell and Baulcombe (1999) Plant J. 20:357-362, and U.S.Pat. No. 6,646,805, each of which is herein incorporated by reference.

vi. Ribozymes

In some embodiments, the polynucleotide expressed by the expressioncassette of the invention is catalytic RNA or has ribozyme activityspecific for the messenger RNA of the SCW polypeptide. Thus, thepolynucleotide causes the degradation of the endogenous messenger RNA,resulting in reduced expression of the SCW polypeptide. This method isdescribed, for example, in U.S. Pat. No. 4,987,071, herein incorporatedby reference.

vii. Small Interfering RNA or Micro RNA

In some embodiments of the invention, inhibition of the expression of aSCW polypeptide may be obtained by RNA interference by expression of agene encoding a micro RNA (miRNA). miRNAs are regulatory agentsconsisting of about 22 ribonucleotides. miRNA are highly efficient atinhibiting the expression of the inhibition of molecular pathways byexpression and binding of antibodies to proteins in plant cells are wellknown in the art. See, for example, Conrad and Sonnewald (2003) NatureBiotech. 21:35-36, incorporated herein by reference.

4. Gene Disruption

In some embodiments of the present invention, the activity of an SCWpolypeptide is reduced or eliminated by disrupting the gene encoding theSCW polypeptide. The gene encoding the SCW polypeptide may be disruptedby any method known in the art. For example, in one embodiment, the geneis disrupted by transposon tagging. In another embodiment, the gene isdisrupted by mutagenizing plants using random or targeted mutagenesis,and selecting for plants that have reduced secondary cell walldevelopment activity.

i. Transposon Tagging

In one embodiment of the invention, transposon tagging is used to reduceor eliminate the SCW activity of one or more SCW polypeptide. Transposontagging comprises inserting a transposon within an endogenous SCW geneto reduce or eliminate expression of the SCW polypeptide. “SCW gene” isintended to mean the gene that encodes an SCW polypeptide according tothe invention.

In this embodiment, the expression of one or more SCW polypeptide isreduced or eliminated by inserting a transposon within a regulatoryregion or coding region of the gene encoding the SCW polypeptide. Atransposon that is within an exon, intron, 5′ or 3′ untranslatedsequence, a promoter, or any other regulatory sequence of a SCW gene maybe used to reduce or eliminate the expression and/or activity of theencoded SCW polypeptide.

Methods for the transposon tagging of specific genes in plants are wellknown in the art. See, for example, Maes, et al., (1999) Trends PlantSci. 4:90-96; Dharmapuri and Sonti (1999) FEMS Microbiol. Lett.179:53-59; Meissner, et al., (2000) Plant J. 22:265-274; Phogat, et al.,(2000) J. Biosci. 25:57-63; Walbot (2000) Curr. Opin. Plant Biol.2:103-107; Gai, et al., (2000) Nucleic Acids Res. 28:94-96; Fitzmaurice,et al., (1999) Genetics 153:1919-1928). In addition, the TUSC processfor selecting Mu insertions in selected genes has been described inBensen, et al., (1995) Plant Cell 7:75-84; Mena, et al., (1996) Science274:1537-1540; and U.S. Pat. No. 5,962,764; each of which is hereinincorporated by reference.

ii. Mutant Plants with Reduced Activity

Additional methods for decreasing or eliminating the expression ofendogenous genes in plants are also known in the art and can besimilarly applied to the instant invention. These methods include otherforms of mutagenesis, such as ethyl methanesulfonate-inducedmutagenesis, deletion mutagenesis, and fast neutron deletion mutagenesisused in a reverse genetics sense (with PCR) to identify plant lines inwhich the endogenous gene has been deleted. For examples of thesemethods see, Ohshima, et al., (1998) Virology 243:472-481; Okubara, etal., (1994) Genetics 137:867-874; and Quesada, et al., (2000) Genetics154:421-436; each of which is herein incorporated by reference. Inaddition, a fast and automatable method for screening for chemicallyinduced mutations, TILLING (Targeting Induced Local Lesions In Genomes),using denaturing HPLC or selective endonuclease digestion of selectedPCR products is also applicable to the instant invention. See, McCallum,et al., (2000) Nat. Biotechnol. 18:455-457, herein incorporated byreference.

Mutations that impact gene expression or that interfere with thefunction (secondary cell wall formation activity) of the encoded proteinare well known in the art. Insertional mutations in gene exons usuallyresult in null-mutants. Mutations in conserved residues are particularlyeffective in inhibiting the secondary cell wall formation activity ofthe encoded protein. Conserved residues of plant SCW polypeptidessuitable for mutagenesis with the goal to eliminate secondary cell walldevelopment activity have been described. Such mutants can be isolatedaccording to well-known procedures, and mutations in different SCW locican be stacked by genetic crossing. See, for example, Gruis, et al.,(2002) Plant Cell 14:2863-2882.

In another embodiment of this invention, dominant mutants can be used totrigger RNA silencing due to gene inversion and recombination of aduplicated gene locus. See, for example, Kusaba, et al., (2003) PlantCell 15:1455-1467.

The invention encompasses additional methods for reducing or eliminatingthe activity of one or more SCW polypeptide. Examples of other methodsfor altering or mutating a genomic nucleotide sequence in a plant areknown in the art and include, but are not limited to, the use of RNA:DNAvectors, RNA:DNA mutational vectors, RNA:DNA repair vectors,mixed-duplex oligonucleotides, self-complementary RNA:DNAoligonucleotides, and recombinogenic oligonucleobases. Such vectors andmethods of use are known in the art. See, for example, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984;each of which are herein incorporated by reference. See also, WO98/49350, WO 99/07865, WO 99/25821, and Beetham, et al., (1999) Proc.Natl. Acad. Sci. USA 96:8774-8778; each of which is herein incorporatedby reference.

iii. Modulating Plant Growth and/or Secondary Cell Wall DevelopmentActivity

In specific methods, the level and/or activity of a secondary cell walldevelopment gene in a plant is increased by increasing the level oractivity of the SCW polypeptide in the plant. Methods for increasing thelevel and/or activity of SCW polypeptides in a plant are discussedelsewhere herein. Briefly, such methods comprise providing a SCWpolypeptide of the invention to a plant and thereby increasing the leveland/or activity of the SCW polypeptide. In other embodiments, an SCWnucleotide sequence encoding an SCW polypeptide can be provided byintroducing into the plant a polynucleotide comprising an SCW nucleotidesequence of the invention, expressing the SCW sequence, increasing theactivity of the SCW polypeptide, and thereby increasing the secondarycell wall formation in the plant or plant part. In other embodiments,the SCW nucleotide construct introduced into the plant is stablyincorporated into the genome of the plant.

In other methods, the number of cells and biomass of a plant tissue isincreased by increasing the level and/or activity of the SCW polypeptidein the plant. Such methods are disclosed in detail elsewhere herein. Inone such method, an SCW nucleotide sequence is introduced into the plantand expression of said SCW nucleotide sequence decreases the activity ofthe SCW polypeptide, and thereby increasing the plant growth and/orsecondary cell wall development in the plant or plant part. In otherembodiments, the SCW nucleotide construct introduced into the plant isstably incorporated into the genome of the plant.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate the level/activity of a plant growth and/or secondarycell wall development polynucleotide and polypeptide in the plant.Exemplary promoters for this embodiment have been disclosed elsewhereherein.

Accordingly, the present invention further provides plants having amodified plant growth and/or secondary cell wall development whencompared to the plant growth and/or secondary cell wall development of acontrol plant tissue. In one embodiment, the plant of the invention hasan increased level/activity of the SCW polypeptide of the invention andthus has increased plant growth and/or secondary cell wall developmentin the plant tissue. In other embodiments, the plant of the inventionhas a reduced or eliminated level of the SCW polypeptide of theinvention and thus has decreased plant growth and/or secondary cell walldevelopment in the plant tissue. In other embodiments, such plants havestably incorporated into their genome a nucleic acid molecule comprisinga SCW nucleotide sequence of the invention operably linked to a promoterthat drives expression in the plant cell.

iv. Modulating Root Development

Methods for modulating root development in a plant are provided. By“modulating root development” is intended any alteration in thedevelopment of the plant root when compared to a control plant. Suchalterations in root development include, but are not limited to,alterations in the growth rate of the primary root, the fresh rootweight, the extent of lateral and adventitious root formation, thevasculature system, meristem development, or radial expansion. Inparticular, the most desirable outcome would be a root with a strongervasculature that improves the standability of the plant and thus reducesroot lodging as well as being less susceptible to pests.

Methods for modulating root development in a plant are provided. Themethods comprise modulating the level and/or activity of the SCWpolypeptide in the plant. In one method, an SCW sequence of theinvention is provided to the plant. In another method, the SCWnucleotide sequence is provided by introducing into the plant apolynucleotide comprising an SCW nucleotide sequence of the invention,expressing the SCW sequence, and thereby modifying root development. Instill other methods, the SCW nucleotide construct introduced into theplant is stably incorporated into the genome of the plant.

In other methods, root development is modulated by altering the level oractivity of the SCW polypeptide in the plant. An increase in SCWactivity can result in at least one or more of the following alterationsto root development, including, but not limited to, larger rootmeristems, increased in root growth, enhanced radial expansion, anenhanced vasculature system, increased root branching, more adventitiousroots, and/or an increase in fresh root weight when compared to acontrol plant.

As used herein, “root growth” encompasses all aspects of growth of thedifferent parts that make up the root system at different stages of itsdevelopment in both monocotyledonous and dicotyledonous plants. It is tobe understood that enhanced root growth can result from enhanced growthof one or more of its parts including the primary root, lateral roots,adventitious roots, etc.

Methods of measuring such developmental alterations in the root systemare known in the art. See, for example, US Patent Application Number2003/0074698 and Werner, et al., (2001) PNAS 18:10487-10492, both ofwhich are herein incorporated by reference.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate root development in the plant. Exemplary promotersfor this embodiment include constitutive promoters and root-preferredpromoters. Exemplary root-preferred promoters have been disclosedelsewhere herein.

Stimulating root growth and increasing root mass by increasing theactivity and/or level of the SCW polypeptide also finds use in improvingthe standability of a plant. The term “resistance to lodging” or“standability” refers to the ability of a plant to fix itself to thesoil. For plants with an erect or semi-erect growth habit, this termalso refers to the ability to maintain an upright position under adverse(environmental) conditions. This trait relates to the size, depth andmorphology of the root system. In addition, stimulating root growth andincreasing root mass by increasing the level and/or activity of the SCWpolypeptide also finds use in promoting in vitro propagation ofexplants.

Furthermore, higher root biomass production due to an increased leveland/or activity of SCW activity has a direct effect on the yield and anindirect effect of production of compounds produced by root cells ortransgenic root cells or cell cultures of said transgenic root cells.One example of an interesting compound produced in root cultures isshikonin, the yield of which can be advantageously enhanced by saidmethods.

Accordingly, the present invention further provides plants havingmodulated root development when compared to the root development of acontrol plant. In some embodiments, the plant of the invention has anincreased level/activity of the SCW polypeptide of the invention and hasenhanced root growth and/or root biomass. In other embodiments, suchplants have stably incorporated into their genome a nucleic acidmolecule comprising a SCW nucleotide sequence of the invention operablylinked to a promoter that drives expression in the plant cell.

v. Modulating Shoot and Leaf Development

Methods are also provided for modulating shoot and leaf development in aplant. By “modulating shoot and/or leaf development” is intended anyalteration in the development of the plant shoot and/or leaf. Suchalterations in shoot and/or leaf development include, but are notlimited to, alterations in shoot meristem development, in leaf number,leaf size, leaf and stem vasculature, internode length, and leafsenescence. As used herein, “leaf development” and “shoot development”encompasses all aspects of growth of the different parts that make upthe leaf system and the shoot system, respectively, at different stagesof their development, both in monocotyledonous and dicotyledonousplants. Methods for measuring such developmental alterations in theshoot and leaf system are known in the art. See, for example, Werner, etal., (2001) PNAS 98:10487-10492 and US Patent Application Number2003/0074698, each of which is herein incorporated by reference.

The method for modulating shoot and/or leaf development in a plantcomprises modulating the activity and/or level of an SCW polypeptide ofthe invention. In one embodiment, an SCW sequence of the invention isprovided. In other embodiments, the SCW nucleotide sequence can beprovided by introducing into the plant a polynucleotide comprising anSCW nucleotide sequence of the invention, expressing the SCW sequence,and thereby modifying shoot and/or leaf development. In otherembodiments, the SCW nucleotide construct introduced into the plant isstably incorporated into the genome of the plant.

In specific embodiments, shoot or leaf development is modulated bydecreasing the level and/or activity of the SCW polypeptide in theplant. An decrease in SCW activity can result in at least one or more ofthe following alterations in shoot and/or leaf development, including,but not limited to, reduced leaf number, reduced leaf surface, reducedvascular, shorter internodes and stunted growth, and retarded leafsenescence, when compared to a control plant.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate shoot and leaf development of the plant. Exemplarypromoters for this embodiment include constitutive promoters,shoot-preferred promoters, shoot meristem-preferred promoters, andleaf-preferred promoters. Exemplary promoters have been disclosedelsewhere herein.

Decreasing SCW activity and/or level in a plant results in shorterinternodes and stunted growth. Thus, the methods of the invention finduse in producing dwarf plants. In addition, as discussed above,modulation of SCW activity in the plant modulates both root and shootgrowth. Thus, the present invention further provides methods foraltering the root/shoot ratio. Shoot or leaf development can further bemodulated by decreasing the level and/or activity of the SCW polypeptidein the plant.

Accordingly, the present invention further provides plants havingmodulated shoot and/or leaf development when compared to a controlplant. In some embodiments, the plant of the invention has an increasedlevel/activity of the SCW polypeptide of the invention, altering theshoot and/or leaf development. Such alterations include, but are notlimited to, increased leaf number, increased leaf surface, increasedvascularity, longer internodes and increased plant stature, as well asalterations in leaf senescence, as compared to a control plant. In otherembodiments, the plant of the invention has a decreased level/activityof the SCW polypeptide of the invention.

vi. Modulating Reproductive Tissue Development

Methods for modulating reproductive tissue development are provided. Inone embodiment, methods are provided to modulate floral development in aplant. By “modulating floral development” is intended any alteration ina structure of a plant's reproductive tissue as compared to a controlplant in which the activity or level of the SCW polypeptide has not beenmodulated. “Modulating floral development” further includes anyalteration in the timing of the development of a plant's reproductivetissue (i.e., a delayed or an accelerated timing of floral development)when compared to a control plant in which the activity or level of theSCW polypeptide has not been modulated. Macroscopic alterations mayinclude changes in size, shape, number, or location of reproductivetissues, the developmental time period that these structures form, orthe ability to maintain or proceed through the flowering process intimes of environmental stress. Microscopic alterations may includechanges to the types or shapes of cells that make up the reproductivetissues.

The method for modulating floral development in a plant comprisesmodulating SCW activity in a plant. In one method, an SCW sequence ofthe invention is provided. An SCW nucleotide sequence can be provided byintroducing into the plant a polynucleotide comprising an SCW nucleotidesequence of the invention, expressing the SCW sequence, and therebymodifying floral development. In other embodiments, the SCW nucleotideconstruct introduced into the plant is stably incorporated into thegenome of the plant.

In specific methods, floral development is modulated by decreasing thelevel or activity of the SCW polypeptide in the plant. A decrease in SCWactivity can result in at least one or more of the following alterationsin floral development, including, but not limited to, retardedflowering, reduced number of flowers, partial male sterility, andreduced seed set, when compared to a control plant. Inducing delayedflowering or inhibiting flowering can be used to enhance yield in foragecrops such as alfalfa. Methods for measuring such developmentalalterations in floral development are known in the art. See, forexample, Mouradov, et al., (2002) The Plant Cell S111-S130, hereinincorporated by reference.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate floral development of the plant. Exemplary promotersfor this embodiment include constitutive promoters, inducible promoters,shoot-preferred promoters, and inflorescence-preferred promoters.

In other methods, floral development is modulated by increasing thelevel and/or activity of the SCW sequence of the invention. Such methodscan comprise introducing an SCW nucleotide sequence into the plant andincreasing the activity of the SCW polypeptide. In other methods, theSCW nucleotide construct introduced into the plant is stablyincorporated into the genome of the plant. Increasing expression of theSCW sequence of the invention can modulate floral development duringperiods of stress. Such methods are described elsewhere herein.Accordingly, the present invention further provides plants havingmodulated floral development when compared to the floral development ofa control plant. Compositions include plants having an increasedlevel/activity of the SCW polypeptide of the invention and having analtered floral development. Compositions also include plants having anincreased level/activity of the SCW polypeptide of the invention whereinthe plant maintains or proceeds through the flowering process in timesof stress.

Methods are also provided for the use of the SCW sequences of theinvention to increase seed size and/or weight. The method comprisesincreasing the activity of the SCW sequences in a plant or plant part,such as the seed. An increase in seed size and/or weight comprises anincreased size or weight of the seed and/or an increase in the size orweight of one or more seed part including, for example, the embryo,endosperm, seed coat, aleurone, or cotyledon.

As discussed above, one of skill will recognize the appropriate promoterto use to increase seed size and/or seed weight. Exemplary promoters ofthis embodiment include constitutive promoters, inducible promoters,seed-preferred promoters, embryo-preferred promoters, andendosperm-preferred promoters.

The method for decreasing seed size and/or seed weight in a plantcomprises decreasing SCW activity in the plant. In one embodiment, theSCW nucleotide sequence can be provided by introducing into the plant apolynucleotide comprising a SCW nucleotide sequence of the invention,expressing the SCW sequence, and thereby increasing seed weight and/orsize. In other embodiments, the SCW nucleotide construct introduced intothe plant is stably incorporated into the genome of the plant.

It is further recognized that increasing seed size and/or weight canalso be accompanied by an increase in the speed of growth of seedlingsor an increase in early vigor. As used herein, the term “early vigor”refers to the ability of a plant to grow rapidly during earlydevelopment, and relates to the successful establishment, aftergermination, of a well-developed root system and a well-developedphotosynthetic apparatus. In addition, an increase in seed size and/orweight can also result in an increase in plant yield when compared to acontrol.

Accordingly, the present invention further provides plants having anincreased seed weight and/or seed size when compared to a control plant.In other embodiments, plants having an increased vigor and plant yieldare also provided. In some embodiments, the plant of the invention hasan increased level/activity of the SCW polypeptide of the invention andhas an increased seed weight and/or seed size. In other embodiments,such plants have stably incorporated into their genome a nucleic acidmolecule comprising a SCW nucleotide sequence of the invention operablylinked to a promoter that drives expression in the plant cell.

vii. Method of Use for SCW Promoter Polynucleotides

The polynucleotides comprising the SCW promoters disclosed in thepresent invention, as well as variants and fragments thereof, are usefulin the genetic manipulation of any host cell, preferably plant cell,when assembled with a DNA construct such that the promoter sequence isoperably linked to a nucleotide sequence comprising a polynucleotide ofinterest. In this manner, the SCW promoter polynucleotides of theinvention are provided in expression cassettes along with apolynucleotide sequence of interest for expression in the host cell ofinterest. The SCW promoter sequences of the invention are expressed in avariety of tissues containing cells that have secondary walls and thusthe promoter sequences can find use in regulating the temporal and/orthe spatial expression of polynucleotides of interest particularly inthe secondary wall-containing cells.

Synthetic hybrid promoter regions are known in the art. Such regionscomprise upstream promoter elements of one polynucleotide operablylinked to the promoter element of another polynucleotide. In anembodiment of the invention, heterologous sequence expression iscontrolled by a synthetic hybrid promoter comprising the SCW promotersequences of the invention, or a variant or fragment thereof, operablylinked to upstream promoter element(s) from a heterologous promoter.Upstream promoter elements that are involved in the plant defense systemhave been identified and may be used to generate a synthetic promoter.See, for example, Rushton, et al., (1998) Curr. Opin. Plant Biol.1:311-315. Alternatively, a synthetic SCW promoter sequence may compriseduplications of the upstream promoter elements found within the SCWpromoter sequences.

It is recognized that the promoter sequence of the invention may be usedwith its native SCW coding sequences. A DNA construct comprising the SCWpromoter operably linked with its native SCW gene may be used totransform any plant of interest to bring about a desired phenotypicchange, such as modulating cell number, modulating root, shoot, leaf,floral, and embryo development, stress tolerance, and any otherphenotype described elsewhere herein.

The promoter nucleotide sequences and methods disclosed herein areuseful in regulating expression of any heterologous nucleotide sequencein a host plant in order to vary the phenotype of a plant. Variouschanges in phenotype are of interest including modifying the fatty acidcomposition in a plant, altering the amino acid content of a plant,altering a plant's pathogen defense mechanism, and the like. Theseresults can be achieved by providing expression of heterologous productsor increased expression of endogenous products in plants. Alternatively,the results can be achieved by providing for a reduction of expressionof one or more endogenous products, particularly enzymes or cofactors inthe plant. These changes result in a change in phenotype of thetransformed plant.

Genes of interest are reflective of the commercial markets and interestsof those involved in the development of the crop. Crops and markets ofinterest change, and as developing nations open up world markets, newcrops and technologies will emerge also. In addition, as ourunderstanding of agronomic traits and characteristics such as yield andheterosis increase, the choice of genes for transformation will changeaccordingly. General categories of genes of interest include, forexample, those genes involved in information, such as zinc fingers,those involved in communication, such as kinases, and those involved inhousekeeping, such as heat shock proteins. More specific categories oftransgenes, for example, include genes encoding important traits foragronomics, insect resistance, disease resistance, herbicide resistance,sterility, grain characteristics, and commercial products. Genes ofinterest include, generally, those involved in oil, starch,carbohydrate, or nutrient metabolism as well as those affecting kernelsize, sucrose loading, and the like.

In certain embodiments the nucleic acid sequences of the presentinvention can be used in combination (“stacked”) with otherpolynucleotide sequences of interest in order to create plants with adesired phenotype. The combinations generated can include multiplecopies of any one or more of the polynucleotides of interest. Thepolynucleotides of the present invention may be stacked with any gene orcombination of genes to produce plants with a variety of desired traitcombinations, including but not limited to traits desirable for animalfeed such as high oil genes (e.g., U.S. Pat. No. 6,232,529); balancedamino acids (e.g., hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801;5,885,802; and 5,703,409); barley high lysine (Williamson, et al.,(1987) Eur. J. Biochem. 165:99-106; and WO 98/20122); and highmethionine proteins (Pedersen, et al., (1986) J. Biol. Chem. 261:6279;Kirihara, et al., (1988) Gene 71:359; and Musumura, et al., (1989) PlantMol. Biol. 12:123)); increased digestibility (e.g., modified storageproteins (U.S. patent application Ser. No. 10/053,410, filed Nov. 7,2001); and thioredoxins (U.S. patent application Ser. No. 10/005,429,filed Dec. 3, 2001)), the disclosures of which are herein incorporatedby reference. The polynucleotides of the present invention can also bestacked with traits desirable for insect, disease or herbicideresistance (e.g., Bacillus thuringiensis toxic proteins (U.S. Pat. Nos.5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser, et al.,(1986) Gene 48:109); lectins (Van Damme, et al., (1994) Plant Mol. Biol.24:825); fumonisin detoxification genes (U.S. Pat. No. 5,792,931);avirulence and disease resistance genes (Jones, et al., (1994) Science266:789; Martin, et al., (1993) Science 262:1432; Mindrinos, et al.,(1994) Cell 78:1089); acetolactate synthase (ALS) mutants that lead toherbicide resistance such as the S4 and/or Hra mutations; inhibitors ofglutamine synthase such as phosphinothricin or basta (e.g., bar gene);and glyphosate resistance (EPSPS gene)); and traits desirable forprocessing or process products such as high oil (e.g., U.S. Pat. No.6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat.No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPGpyrophosphorylases (AGPase), starch synthases (SS), starch branchingenzymes (SBE) and starch debranching enzymes (SDBE)); and polymers orbioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase,polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert,et al., (1988) J. Bacteriol. 170:5837-5847) facilitate expression ofpolyhydroxyalkanoates (PHAs)), the disclosures of which are hereinincorporated by reference. One could also combine the polynucleotides ofthe present invention with polynucleotides affecting agronomic traitssuch as male sterility (e.g., see, U.S. Pat. No. 5,583,210), stalkstrength, flowering time, or transformation technology traits such ascell cycle regulation or gene targeting (e.g., WO 99/61619; WO 00/17364;WO 99/25821), the disclosures of which are herein incorporated byreference.

In one embodiment, sequences of interest improve plant growth and/orcrop yields. For example, sequences of interest include agronomicallyimportant genes that result in improved primary or lateral root systems.Such genes include, but are not limited to, nutrient/water transportersand growth induces. Examples of such genes, include but are not limitedto, maize plasma membrane H⁺-ATPase (MHA2) (Frias, et al., (1996) PlantCell 8:1533-44); AKT1, a component of the potassium uptake apparatus inArabidopsis, (Spalding, et al., (1999) J Gen Physiol 113:909-18); RMLgenes which activate cell division cycle in the root apical cells(Cheng, et al., (1995) Plant Physiol 108:881); maize glutaminesynthetase genes (Sukanya, et al., (1994) Plant Mol Biol 26:1935-46) andhemoglobin (Duff, et al., (1997) J. Biol. Chem. 27:16749-16752,Arredondo-Peter, et al., (1997) Plant Physiol. 115:1259-1266;Arredondo-Peter, et al., (1997) Plant Physiol 114:493-500 and referencessited therein). The sequence of interest may also be useful inexpressing antisense nucleotide sequences of genes that that negativelyaffects root development.

Additional, agronomically important traits such as oil, starch, andprotein content can be genetically altered in addition to usingtraditional breeding methods. Modifications include increasing contentof oleic acid, saturated and unsaturated oils, increasing levels oflysine and sulfur, providing essential amino acids, and alsomodification of starch. Hordothionin protein modifications are describedin U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389, hereinincorporated by reference. Another example is lysine and/or sulfur richseed protein encoded by the soybean 2S albumin described in U.S. Pat.No. 5,850,016, and the chymotrypsin inhibitor from barley, described inWilliamson, et al., (1987) Eur. J. Biochem. 165:99-106, the disclosuresof which are herein incorporated by reference.

Derivatives of the coding sequences can be made by site-directedmutagenesis to increase the level of preselected amino acids in theencoded polypeptide. For example, the gene encoding the barley highlysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor,U.S. patent application Ser. No. 08/740,682, filed Nov. 1, 1996, and WO98/20133, the disclosures of which are herein incorporated by reference.Other proteins include methionine-rich plant proteins such as fromsunflower seed (Lilley, et al., (1989) Proceedings of the World Congresson Vegetable Protein Utilization in Human Foods and Animal Feedstuffs,ed. Applewhite (American Oil Chemists Society, Champaign, Ill.), pp.497-502; herein incorporated by reference); corn (Pedersen, et al.,(1986) J. Biol. Chem. 261:6279; Kirihara, et al., (1988) Gene 71:359;both of which are herein incorporated by reference); and rice (Musumura,et al., (1989) Plant Mol. Biol. 12:123, herein incorporated byreference). Other agronomically important genes encode latex, Floury 2,growth factors, seed storage factors, and transcription factors.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European Corn Borer, and the like.Such genes include, for example, Bacillus thuringiensis toxic proteingenes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756;5,593,881; and Geiser, et al., (1986) Gene 48:109); and the like.

Genes encoding disease resistance traits include detoxification genes,such as against fumonosin (U.S. Pat. No. 5,792,931); avirulence (avr)and disease resistance (R) genes (Jones, et al., (1994) Science 266:789;Martin, et al., (1993) Science 262:1432; and Mindrinos, et al., (1994)Cell 78:1089); and the like.

Herbicide resistance traits may include genes coding for resistance toherbicides that act to inhibit the action of acetolactate synthase(ALS), in particular the sulfonylurea-type herbicides (e.g., theacetolactate synthase (ALS) gene containing mutations leading to suchresistance, in particular the S4 and/or Hra mutations), genes coding forresistance to herbicides that act to inhibit action of glutaminesynthase, such as phosphinothricin or basta (e.g., the bar gene), orother such genes known in the art. The bar gene encodes resistance tothe herbicide basta, the nptII gene encodes resistance to theantibiotics kanamycin and geneticin, and the ALS-gene mutants encoderesistance to the herbicide chlorsulfuron.

Sterility genes can also be encoded in an expression cassette andprovide an alternative to physical detasseling. Examples of genes usedin such ways include male tissue-preferred genes and genes with malesterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210.Other genes include kinases and those encoding compounds toxic to eithermale or female gametophytic development.

The quality of grain is reflected in traits such as levels and types ofoils, saturated and unsaturated, quality and quantity of essential aminoacids, and levels of cellulose. In corn, modified hordothionin proteinsare described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and5,990,389.

Commercial traits can also be encoded on a gene or genes that couldincrease for example, starch for ethanol production, or provideexpression of proteins. Another important commercial use of transformedplants is the production of polymers and bioplastics such as describedin U.S. Pat. No. 5,602,321. Genes such as β-Ketothiolase, PHBase(polyhydroxybutyrate synthase), and acetoacetyl-CoA reductase (see,Schubert, et al., (1988) J. Bacteriol. 170:5837-5847) facilitateexpression of polyhydroxyalkanoates (PHAs).

Exogenous products include plant enzymes and products as well as thosefrom other sources including procaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones, and the like. The levelof proteins, particularly modified proteins having improved amino aciddistribution to improve the nutrient value of the plant, can beincreased. This is achieved by the expression of such proteins havingenhanced amino acid content.

This invention can be better understood by reference to the followingnon-limiting examples. It will be appreciated by those skilled in theart that other embodiments of the invention may be practiced withoutdeparting from the spirit and the scope of the invention as hereindisclosed and claimed.

EXAMPLES Example 1 Isolation of SCW Sequences

Of the genes identified thus for to be involved in plant cell wallformation in maize, the cellulose synthase (CesA) genes, CesA10, CesA11and CesA12 along with another gene, brittle stalk-2 (Bk2), are ofparticular interest because of their involvement in secondary cell wallformation (Appenzeller, et al., (2004) “Cellulose synthesis in maize:isolation and expression analysis of the cellulose synthase (CesA) genefamily”; Cellulose 11:287-299; Ching, et al., (2006) “Brittle stalk 2encodes a putative glycosylphosphatidylinositol-anchored protein thataffects mechanical strength of maize tissues by altering the compositionand structure of secondary cell walls”; Planta 224:1174-1184). As aresult, we used these four genes as a reference set with the objectiveof identifying other genes the expression patterns of which are stronglyassociated with those of the reference genes. The genes identified withthis approach are believed to perform important roles in secondary cellwall formation. FIG. 3 shows a summary of the expression patterns ofthese four reference genes across 12 key tissue types of maize. Note thecharacteristic expressions in stalk tissue, which is a key site ofsecondary cell wall formation in maize.

Example 2 Identification of Genes with Strongly Correlated Expression toReference Genes

Curation of these genes relied on the similarity of their expressionpatterns to those from the reference set across a large number librariesin the MPSS™ data set. Pioneer-DuPont has an extensive, proprietarycollection of 227 maize tissue/treatment MPSS™ samples that cover a widerange of plant structure and biology. The MPSS samples' data is arrayedin a large table against with correlation analyses can be performed.Pearson's correlation coefficients were calculated across 227 samplesfor pairs in a way that each pair consisted of one member from thereference and the second member from the remaining tags. In doing so alist of four R and R² values for each subject tag hit was generated, oneto each of the four reference genes (specifically to the tag for thosegenes). These four values were then averaged and ranked in descendingorder. Those at the top of the list will have the expression patternacross the maize MPSS sample set that is most similar to the fourreference genes. For purposes of this study, a minimum cutoff average Rvalue of approximately 0.7 was established. There were additional tagsthat had correlations below this, but those above this threshold werechosen for continued analysis.

Example 3 Curation of Information about Correlated Genes

The high scoring MPSS tags were mapped to their respective genes ororigins using proprietary and public genomic and transcript sequenceresources. Some MPSS tags proved to be alternate tags for the referencegenes, and were thus excluded. Where there were ambiguous tag-to-genematches, the gene of interest was revealed by conferring with otherexpression data, such as from ESTs, wherein expression in stalk tissueespecially was considered evidence of the correct gene. The bestdescription of the gene and its gene product, identifying promoters,transcript regions, ORFs, and conceptual translations were thusobtained. This often required manual curations. Then, the map positionsof the genes were also determined, primarily using the proprietary PHDv1.2 map. The conceptual translations of the genes were re-analyzed todiscern their respective likely functions. The Swiss-Prot database andvarious computational tools and other resources were used for thispurpose.

Example 4 Validation of Gene Discovery Candidates Using Reverse Genetics

The secondary wall forming genes (SCW) datasheet also shows the currentprioritizations made for validation by selected knockout mutagenesis(reverse genetics). Reverse genetics resources enable the functionalanalysis of sequenced gene candidates. Pioneer's TUSC system is alibrary of insertion mutants created with the maize transposable elementfamily, Mutator, Meeley and Briggs (1995) “Reverse genetics for maize”Maize Genet. Coop. Newsl. 69:67-82. This is a patented approach withwhich to identify transposon insertion mutants for a selected genesequence (Briggs and Meeley, U.S. Pat. No. 5,962,764).

In order to characterize the candidate genes, a working gene model foreach prioritized SCW candidate would be translated into the bestavailable genomic sequence model by making use of available maizegenomic data from public and proprietary sequence databases.Gene-specific PCR primers are then designed for each candidate sequenceto be submitted through the TUSC screening process. These primers wouldbe validated in pairs on non-mutant genomic DNA, and each primer is thensubsequently paired together with a universal Mutator-specific primer initerative insertion screens against DNA from the TUSC population.Individual insertion alleles identified in this procedure are confirmedfor heritability among F2 progeny from selected TUSC lines, and seedfrom each positive line is retrieved for field propagation. The fieldmaterials define segregating maize families in which insertion (null)alleles for SCW genes are genetically segregating. Mutator insertionpositions are then be determined by PCR and DNA sequencing. Genetically,each mutant allele would be submitted to several rounds of backcrossingto clean up background mutations, and subsequently selfed to createsegregating mutant families and homozygous SCW gene mutant stocks indefined genetic backgrounds. Interallelic crosses can also be attemptedwhenever possible to help speed the phenotypic analysis of selectedmutant loci. Phenotypic analysis of SCW components proceeds by comparingnull mutants with their appropriate controls, and the top candidatesthen are advanced for more detailed studies on the contribution of eachcandidate gene to SCW formation/deposition. Several tests could beemployed, such as internodal mechanical strength, celluloseconcentration in the stalk walls, and perhaps scanning electronmicroscopy, to determine whether the integrity of the secondary wall hasbeen affected (Appenzeller, et al., (2004) “Cellulose synthesis inmaize: isolation and expression analysis of the cellulose synthase(CesA) gene family”; Cellulose 11:287-299; Ching, et al., (2006)“Brittle stalk 2 encodes a putativeglycosylphosphatidylinositol-anchored protein that affects mechanicalstrength of maize tissues by altering the composition and structure ofsecondary cell walls”; Planta 224:1174-1184).

Example 5 Transformation and Regeneration of Transgenic Plants

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing the ZmSCW sequence operably linked to thedrought-inducible promoter RAB17 promoter (Vilardell, et al., (1990)Plant Mol Biol 14:423-432) and the selectable marker gene PAT, whichconfers resistance to the herbicide Bialaphos. Alternatively, theselectable marker gene is provided on a separate plasmid. Transformationis performed as follows. Media recipes follow below.

Preparation of Target Tissue:

The ears are husked and surface sterilized in 30% Clorox bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5-cm target zone in preparation forbombardment.

Preparation of DNA:

A plasmid vector comprising the SCW sequence operably linked to anubiquitin promoter is made. This plasmid DNA plus plasmid DNA containinga PAT selectable marker is precipitated onto 1.1 μm (average diameter)tungsten pellets using a CaCl₂ precipitation procedure as follows:

100 μl prepared tungsten particles in water

10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)

100 μl 12.5 M CaCl₂

10 μl 0.1 M spermidine

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 ml 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

Particle Gun Treatment:

The sample plates are bombarded at level #4 in particle gun #HE34-1 or#HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment:

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for increased drought tolerance. Assaysto measure improved drought tolerance are routine in the art andinclude, for example, increased kernel-earring capacity yields underdrought conditions when compared to control maize plants under identicalenvironmental conditions. Alternatively, the transformed plants can bemonitored for a modulation in meristem development (i.e., a decrease inspikelet formation on the ear). See, for example, Bruce, et al., (2002)Journal of Experimental Botany 53:1-13.

Bombardment and Culture Media:

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H₂O); and8.5 mg/l silver nitrate (added after sterilizing the medium and coolingto room temperature). Selection medium (560R) comprises 4.0 g/l N6 basalsalts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511),0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought tovolume with D-I H₂O following adjustment to pH 5.8 with KOH); 3.0 g/lGelrite (added after bringing to volume with D-I H₂O); and 0.85 mg/lsilver nitrate and 3.0 mg/l bialaphos (both added after sterilizing themedium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog (1962)Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/lsucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite (addedafter bringing to volume with D-I H₂O); and 1.0 mg/l indoleacetic acidand 3.0 mg/l bialaphos (added after sterilizing the medium and coolingto 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinicacid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/lglycine brought to volume with polished D-I H₂O), 0.1 g/l myo-inositol,and 40.0 g/l sucrose (brought to volume with polished D-I H₂O afteradjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing tovolume with polished D-I H₂O), sterilized and cooled to 60° C.

Example 6 Agrobacterium-Mediated Transformation

For Agrobacterium-mediated transformation of maize with an antisensesequence of the ZmSCW sequence of the present invention, preferably themethod of Zhao is employed (U.S. Pat. No. 5,981,840, and PCT PatentPublication WO98/32326; the contents of which are hereby incorporated byreference). Briefly, immature embryos are isolated from maize and theembryos contacted with a suspension of Agrobacterium, where the bacteriaare capable of transferring the SCW sequence to at least one cell of atleast one of the immature embryos (step 1: the infection step). In thisstep the immature embryos are preferably immersed in an Agrobacteriumsuspension for the initiation of inoculation. The embryos areco-cultured for a time with the Agrobacterium (step 2: theco-cultivation step). Preferably the immature embryos are cultured onsolid medium following the infection step. Following this co-cultivationperiod an optional “resting” step is contemplated. In this resting step,the embryos are incubated in the presence of at least one antibioticknown to inhibit the growth of Agrobacterium without the addition of aselective agent for plant transformants (step 3: resting step).Preferably the immature embryos are cultured on solid medium withantibiotic, but without a selecting agent, for elimination ofAgrobacterium and for a resting phase for the infected cells. Next,inoculated embryos are cultured on medium containing a selective agentand growing transformed callus is recovered (step 4: the selectionstep). Preferably, the immature embryos are cultured on solid mediumwith a selective agent resulting in the selective growth of transformedcells. The callus is then regenerated into plants (step 5: theregeneration step), and preferably calli grown on selective medium arecultured on solid medium to regenerate the plants. Plants are monitoredand scored for a modulation in meristem development. For instance,alterations of size and appearance of the shoot and floral meristemsand/or increased yields of leaves, flowers, and/or fruits.

Example 7 Expression Patterns

Table 3 below shows a list of the secondary cell wall forming genes indescending order according to their average Pearson's correlationcoefficients to the reference genes for expression pattern across 227maize MPSS libraries. In addition, there is a summary statement of thelikely function of each gene and its role in secondary cell wallformation. Many of the genes are implicated in either carbohydrate orlignin metabolism, the two main sub components of the secondary cellwall. However, the list contains many novels genes as well and geneswithout a present direct relationship to either carbohydrate or ligninsynthesis or modification.

TABLE 3 Secondary Cell Wall Forming Genes - Expression Correlations andFunction Table 3. Secondary Wall Formation Genes - ExpressionCorrelations, Descriptions and Likely Function Expression Correlation(Average Sequence Pearson's R Name Value) Description and LikelyFunction SCW_CMI_04 0.85 Related to plant specific proteins of unknownfunction, but sometimes annotated as ‘leaf senescence relatedprotein-like’ SCW_CMI_06 0.83 Member of a plant specific family ofconserved proteins. Importantly, three distinct members of the familyoccur on short list of SCW associated genes (see CMI_06, CMI_25, andCMI_38). Biochemical function unknown, predicted to have a transmembranedomain. SCW_CMI_50 0.75 Myb transcription factor SCW_CMI_22 0.75Xyloglucan endotransglycosylase/hydrolase protein 8 precursor (EC2.4.1.207). First of type apparently associated with secondary cellwalls SCW_CMI_28 0.73 Fasciclin-like arabinogalactan protein 8precursor; glycosylphosphatidylinositol-anchored protein SCW_CMI_51 0.78Endoglucanase 5 precursor (EC 3.2.1.4) SCW_CMI_48 0.77 Fasciclin-likearabinogalactan protein 8 precursor;glycosylphosphatidylinositol-anchored protein SCW_CMI_54 0.79 Leafsenescence protein-like SCW_CMI_13 0.80 L-ascorbate oxidase precursor(EC 1.10.3.3) or Laccase 4 precursor (EC 1.10.3.2) (Benzenediol:oxygenoxidoreductase). Likely lignin formation related. SCW_CMI_09 0.82L-ascorbate oxidase precursor (EC 1.10.3.3) or Laccase 4 precursor (EC1.10.3.2) (Benzenediol:oxygen oxidoreductase). Likely lignin formationrelated. SCW_CMI_56 0.83 Conserved Plant specific Unknown ProteinSCW_CMI_53 0.83 Conserved Plant specific Unknown Protein SCW_CMI_21 0.76Anthranilate N-hydroxycinnamoyl/benzoyltransferase (EC 2.3.1.144).Likely lignin formation related. SCW_CMI_10 0.82 Putative diseaseresistance response protein-related/dirigent protein. Likely ligninformation related. SCW_CMI_49 0.80 Putative disease resistance responseprotein-related/dirigent protein. Likely lignin formation related.SCW_CMI_47 0.77 Laccase or L-ascorbate oxidase SCW_CMI_45 0.77 ConservedPlant specific Unknown Protein SCW_CMI_55 0.77 Novel intregral membraneprotein; glucose-6-phosphate/phosphate- tranlocator; solute transporterfamily SCW_CMI_46 0.75 Putative lipid binding protein SCW_CMI_23 0.75Naringenin, 2-oxoglutarate 3-dioxygenase; Flavonone-3-hydroxylase.Likely lignin formation related. SCW_CMI_39 0.86 Putative3-methyladenine-DNA glycosylase (EC 3.2.2.20) SCW_CMI_43 0.67 Novelprotein, possibly plant specific, which may bear a ring-H2 zinc fingerdomain. SCW_CMI_57 0.73 Conserved Plant specific Unknown ProteinSCW_CMI_44 0.72 NAC Domain Protein, Arabidopsis related annotated asinvolved in Secondary Cell Wall Formation SCW_CMI_58 0.71 Unknown.Membrane lipoprotein lipid attachment site-containing protein-likeSCW_CMI_26 0.74 Cytochrome P450, or Flavonoid 3′-monooxygenase (see EC1.14.—.— or EC 1.14.13.—). Likely lignin formation related. SCW_CMI_170.80 Novel plant specific presumed membrane attached GPI-Anchoredprotein SCW_CMI_32 0.72 Myb-related transcription factor. Onlytranscription factor on list. Candidate for control of battery ofsecondary cell wall associated genes. SCW_CMI_01 0.86 Possibleglucosyltransferase, may be related to Exostosin-1. SCW_CMI_05 0.84Related to Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase P (EC 2.4.1.135) (Beta-1,3-glucuronyltransferase P) SCW_CMI_40 0.71 Related to plant specificproteins of unknown function, but sometimes annotated as ‘auxin inducedprotein’ SCW_CMI_41 0.66 Novel plant specific protein, possibly bearinga possible BAG domain (Bcl-2 associated anthogene). May function aschaperone.

FIG. 3 shows the expression pattern across twelve key maize tissues ofthe reference secondary cell wall forming genes along with the newsecondary cell wall genes found in this study. Note the markedassociation with stalk expression, a key site of secondary cell wallformation, but also to a lesser extent roots and leaves, where secondarycell walls also form. Interestingly, in apical meristem, where there isessentially only primary cell walls, expression of these genes is nearlyabsent.

Example 8 Role of Cellulose in Mechanical Strength

Cellulose is the largest single chemical constituent in the vegetativebiomass (FIG. 2). It accounts for an average of 50% of the maize stoverdry mass. As a largest constituent, any limitation in its deposition canlimit growth and thus biomass accumulation. Grain yield in maize is afunction of total biomass as the harvest index (ratio of grain to totalaboveground biomass) has stayed around 50% for the last century. Anyincrease in biomass will lead to increased grain yield. The genes thatspecifically affect cellulose formation will help increase carbohydratecontent of the biomass, making it more suitable for silage as well asethanol production. In addition, a stalk with increased celluloseconcentration will have increased mechanical strength, which will makeit less likely to lodge and thus indirectly contribute to grain yieldincrease. The genes for lignin formation and cell wall cross-linkingmight also help increase the strength of the stalk. Lignin, beinghydrophobic, increases the strength of cellulose by excluding water fromaround the latter. Dry cellulose is known to be stronger than moistcellulose.

Leaves with increased cellulose concentration, because they aremechanically stronger, will be able to maintain a more erect phenotype,which will increase photosynthetic capacity per unit land area byreducing shading.

Example 9 Relative Contribution of Maize Stalk Rind and Inner Tissue toDifferent Stalk Characteristics and Mechanical Strength

The data shown in FIG. 1 was derived from seven hybrids grown at threedensities (27, 43 and 59 K per acre) in three replications each in 2001.Two stalks were sampled from each replication. Internodes 3 and 4 belowthe ear were subjected to a 3-point flexural test with an Instron. Aftermeasuring load to cause a break in the internode, the 3^(rd) internodewas separated into rind and inner tissue. Path coefficient analyses wereperformed using rind and inner tissue as independent variables (X₁ andX₂, respectively) and the whole stalk as the dependent variable (Y). Themultiple regression equation would look like: Y=a+b₁X₁+b₂X₂+e where a isthe intercept, b is regression co-efficient, and e error. Pathcoefficients were calculated as follows: pYX_(n)=b_(n)*SX_(n)/sY where nis 1 or 2, p is path co-efficient, and s is standard deviation Thecontribution of each independent variable to whole stalk (Y) wascalculated as follows: s_(YXn)*r_(YXn) where sYXn is covariance of Y andXn, and r is the Pearson correlation coefficient between these twovariables. The unexplained variation for diameter is attributable to thecorn stalk not being perfectly round and the difficulty thus associatedwith determining the cross-sectional area accurately. Some othervariables, like size and number of vascular bundles and their density,may account for the remaining variation in strength.

Selection for a desirable allele or overexpressing the gene thereof, byremoving the limitation of the particular step it encodes the enzyme forcatalyzing, may either lead to an increase in the percentage of thatparticular polysaccharide (composition changed) or of the whole cellwall (composition not changed). In the latter case, the additional drymatter could be accommodated in enlarged vascular bundles which could,in turn, result in an increased diameter

Example 10 Soybean Embryo Transformation

Soybean embryos are bombarded with a plasmid containing an SCW sequenceoperably linked to an ubiquitin promoter as follows. To induce somaticembryos, cotyledons, 3-5 mm in length dissected from surface-sterilized,immature seeds of the soybean cultivar A2872, are cultured in the lightor dark at 26° C. on an appropriate agar medium for six to ten weeks.Somatic embryos producing secondary embryos are then excised and placedinto a suitable liquid medium. After repeated selection for clusters ofsomatic embryos that multiplied as early, globular-staged embryos, thesuspensions are maintained as described below.

Soybean embryogenic suspension cultures can be maintained in 35 mlliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 ml ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein, et al., (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont BiolisticPDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz, et al., (1983) Gene 25:179-188), and the 3′ region of thenopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette comprising an SCW sense sequenceoperably linked to the ubiquitin promoter can be isolated as arestriction fragment. This fragment can then be inserted into a uniquerestriction site of the vector carrying the marker gene.

To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (inorder): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post-bombardment with freshmedia containing 50 mg/ml hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 11 Sunflower Meristem Tissue Transformation

Sunflower meristem tissues are transformed with an expression cassettecontaining an SCW sequence operably linked to a ubiquitin promoter asfollows (see also, European Patent Number EP 0 486233, hereinincorporated by reference, and Malone-Schoneberg, et al., (1994) PlantScience 103:199-207). Mature sunflower seed (Helianthus annulus L.) aredehulled using a single wheat-head thresher. Seeds are surfacesterilized for 30 minutes in a 20% Clorox bleach solution with theaddition of two drops of Tween 20 per 50 ml of solution. The seeds arerinsed twice with sterile distilled water.

Split embryonic axis explants are prepared by a modification ofprocedures described by Schrammeijer, et al., (Schrammeijer, et al.,(1990) Plant Cell Rep. 9:55-60). Seeds are imbibed in distilled waterfor 60 minutes following the surface sterilization procedure. Thecotyledons of each seed are then broken off, producing a clean fractureat the plane of the embryonic axis. Following excision of the root tip,the explants are bisected longitudinally between the primordial leaves.The two halves are placed, cut surface up, on GBA medium consisting ofMurashige and Skoog mineral elements (Murashige, et al., (1962) Physiol.Plant., 15:473-497), Shepard's vitamin additions (Shepard (1980) inEmergent Techniques for the Genetic Improvement of Crops (University ofMinnesota Press, St. Paul, Minn.), 40 mg/l adenine sulfate, 30 g/lsucrose, 0.5 mg/l 6-benzyl-aminopurine (BAP), 0.25 mg/l indole-3-aceticacid (IAA), 0.1 mg/l gibberellic acid (GA₃), pH 5.6, and 8 g/l Phytagar.

The explants are subjected to microprojectile bombardment prior toAgrobacterium treatment (Bidney, et al., (1992) Plant Mol. Biol.18:301-313). Thirty to forty explants are placed in a circle at thecenter of a 60×20 mm plate for this treatment. Approximately 4.7 mg of1.8 mm tungsten microprojectiles are resuspended in 25 ml of sterile TEbuffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0) and 1.5 ml aliquots are usedper bombardment. Each plate is bombarded twice through a 150 mm nytexscreen placed 2 cm above the samples in a PDS 1000® particleacceleration device.

Disarmed Agrobacterium tumefaciens strain EHA105 is used in alltransformation experiments. A binary plasmid vector comprising theexpression cassette that contains the SCW gene operably linked to theubiquitin promoter is introduced into Agrobacterium strain EHA105 viafreeze-thawing as described by Holsters, et al., (1978) Mol. Gen. Genet.163:181-187. This plasmid further comprises a kanamycin selectablemarker gene (i.e, nptII). Bacteria for plant transformation experimentsare grown overnight (28° C. and 100 RPM continuous agitation) in liquidYEP medium (10 gm/l yeast extract, 10 gm/l Bactopeptone, and 5 gm/lNaCl, pH 7.0) with the appropriate antibiotics required for bacterialstrain and binary plasmid maintenance. The suspension is used when itreaches an OD₆₀₀ of about 0.4 to 0.8. The Agrobacterium cells arepelleted and resuspended at a final OD₆₀₀ of 0.5 in an inoculationmedium comprised of 12.5 mM MES pH 5.7, 1 gm/l NH₄Cl, and 0.3 gm/lMgSO₄.

Freshly bombarded explants are placed in an Agrobacterium suspension,mixed, and left undisturbed for 30 minutes. The explants are thentransferred to GBA medium and co-cultivated, cut surface down, at 26° C.and 18-hour days. After three days of co-cultivation, the explants aretransferred to 374B (GBA medium lacking growth regulators and a reducedsucrose level of 1%) supplemented with 250 mg/l cefotaxime and 50 mg/lkanamycin sulfate. The explants are cultured for two to five weeks onselection and then transferred to fresh 374B medium lacking kanamycinfor one to two weeks of continued development. Explants withdifferentiating, antibiotic-resistant areas of growth that have notproduced shoots suitable for excision are transferred to GBA mediumcontaining 250 mg/l cefotaxime for a second 3-day phytohormonetreatment. Leaf samples from green, kanamycin-resistant shoots areassayed for the presence of NPTII by ELISA and for the presence oftransgene expression by assaying for a modulation in meristemdevelopment (i.e., an alteration of size and appearance of shoot andfloral meristems).

NPTII-positive shoots are grafted to Pioneer® hybrid 6440 in vitro-grownsunflower seedling rootstock. Surface sterilized seeds are germinated in48-0 medium (half-strength Murashige and Skoog salts, 0.5% sucrose, 0.3%gelrite, pH 5.6) and grown under conditions described for explantculture. The upper portion of the seedling is removed, a 1 cm verticalslice is made in the hypocotyl, and the transformed shoot inserted intothe cut. The entire area is wrapped with parafilm to secure the shoot.Grafted plants can be transferred to soil following one week of in vitroculture. Grafts in soil are maintained under high humidity conditionsfollowed by a slow acclimatization to the greenhouse environment.Transformed sectors of T₀ plants (parental generation) maturing in thegreenhouse are identified by NPTII ELISA and/or by SCW activity analysisof leaf extracts while transgenic seeds harvested from NPTII-positive T₀plants are identified by SCW activity analysis of small portions of dryseed cotyledon.

An alternative sunflower transformation protocol allows the recovery oftransgenic progeny without the use of chemical selection pressure. Seedsare dehulled and surface-sterilized for 20 minutes in a 20% Cloroxbleach solution with the addition of two to three drops of Tween 20 per100 ml of solution, then rinsed three times with distilled water.Sterilized seeds are imbibed in the dark at 26° C. for 20 hours onfilter paper moistened with water. The cotyledons and root radical areremoved, and the meristem explants are cultured on 374E (GBA mediumconsisting of MS salts, Shepard vitamins, 40 mg/l adenine sulfate, 3%sucrose, 0.5 mg/l 6-BAP, 0.25 mg/l IAA, 0.1 mg/l GA, and 0.8% Phytagarat pH 5.6) for 24 hours under the endogenous genes. See, for example,Javier, et al., (2003) Nature 425:257-263, herein incorporated byreference.

For miRNA interference, the expression cassette is designed to expressan RNA molecule that is modeled on an endogenous miRNA gene. The miRNAgene encodes an RNA that forms a hairpin structure containing a22-nucleotide sequence that is complementary to another endogenous gene(target sequence). For suppression of SCW expression, the 22-nucleotidesequence is selected from a SCW transcript sequence and contains 22nucleotides of said SCW sequence in sense orientation and 21 nucleotidesof a corresponding antisense sequence that is complementary to the sensesequence. miRNA molecules are highly efficient at inhibiting theexpression of endogenous genes, and the RNA interference they induce isinherited by subsequent generations of plants.

2. Polypeptide-Based Inhibition of Gene Expression

In one embodiment, the polynucleotide encodes a zinc finger protein thatbinds to a gene encoding an SCW polypeptide, resulting in reducedexpression of the gene. In particular embodiments, the zinc fingerprotein binds to a regulatory region of an SCW gene. In otherembodiments, the zinc finger protein binds to a messenger RNA encodingan SCW polypeptide and prevents its translation. Methods of selectingsites for targeting by zinc finger proteins have been described, forexample, in U.S. Pat. No. 6,453,242, and methods for using zinc fingerproteins to inhibit the expression of genes in plants are described, forexample, in US Patent Application Publication Number 20030037355; eachof which is herein incorporated by reference.

3. Polypeptide-Based Inhibition of Protein Activity

In some embodiments of the invention, the polynucleotide encodes anantibody that binds to at least one SCW polypeptide, and reduces thesecondary cell wall formation activity of the SCW polypeptide. Inanother embodiment, the binding of the antibody results in increasedturnover of the antibody-SCW complex by cellular quality controlmechanisms. The expression of antibodies in plant cells and dark. Theprimary leaves are removed to expose the apical meristem, around 40explants are placed with the apical dome facing upward in a 2 cm circlein the center of 374M (GBA medium with 1.2% Phytagar), and then culturedon the medium for 24 hours in the dark.

Approximately 18.8 mg of 1.8 μm tungsten particles are resuspended in150 μl absolute ethanol. After sonication, 8 μl of it is dropped on thecenter of the surface of macrocarrier. Each plate is bombarded twicewith 650 psi rupture discs in the first shelf at 26 mm of Hg helium gunvacuum.

The plasmid of interest is introduced into Agrobacterium tumefaciensstrain EHA105 via freeze thawing as described previously. The pellet ofovernight-grown bacteria at 28° C. in a liquid YEP medium (10 g/l yeastextract, 10 g/l Bactopeptone, and 5 g/l NaCl, pH 7.0) in the presence of50 μg/l kanamycin is resuspended in an inoculation medium (12.5 mM 2-mM2-(N-morpholino) ethanesulfonic acid, MES, 1 g/l NH₄Cl and 0.3 g/l MgSO₄at pH 5.7) to reach a final concentration of 4.0 at OD 600.Particle-bombarded explants are transferred to GBA medium (374E), and adroplet of bacteria suspension is placed directly onto the top of themeristem. The explants are co-cultivated on the medium for 4 days, afterwhich the explants are transferred to 374C medium (GBA with 1% sucroseand no BAP, IAA, GA3 and supplemented with 250 μg/ml cefotaxime). Theplantlets are cultured on the medium for about two weeks under 16-hourday and 26° C. incubation conditions.

Explants (around 2 cm long) from two weeks of culture in 374C medium arescreened for a modulation in meristem development (i.e., an alterationof size and appearance of shoot and floral meristems). After positive(i.e., a change in SCW expression) explants are identified, those shootsthat fail to exhibit an alteration in SCW activity are discarded, andevery positive explant is subdivided into nodal explants. One nodalexplant contains at least one potential node. The nodal segments arecultured on GBA medium for three to four days to promote the formationof auxiliary buds from each node. Then they are transferred to 374Cmedium and allowed to develop for an additional four weeks. Developingbuds are separated and cultured for an additional four weeks on 374Cmedium. Pooled leaf samples from each newly recovered shoot are screenedagain by the appropriate protein activity assay. At this time, thepositive shoots recovered from a single node will generally have beenenriched in the transgenic sector detected in the initial assay prior tonodal culture.

Recovered shoots positive for altered SCW expression are grafted toPioneer hybrid 6440 in vitro-grown sunflower seedling rootstock. Therootstocks are prepared in the following manner. Seeds are dehulled andsurface-sterilized for 20 minutes in a 20% Clorox bleach solution withthe addition of two to three drops of Tween 20 per 100 ml of solution,and are rinsed three times with distilled water. The sterilized seedsare germinated on the filter moistened with water for three days, thenthey are transferred into 48 medium (half-strength MS salt, 0.5%sucrose, 0.3% gelrite pH 5.0) and grown at 26° C. under the dark forthree days, then incubated at 16-hour-day culture conditions. The upperportion of selected seedling is removed, a vertical slice is made ineach hypocotyl, and a transformed shoot is inserted into a V-cut. Thecut area is wrapped with parafilm. After one week of culture on themedium, grafted plants are transferred to soil. In the first two weeks,they are maintained under high humidity conditions to acclimatize to agreenhouse environment.

Example 12 Variants of SCW Sequences

A. Variant Nucleotide Sequences of SCW That Do Not Alter the EncodedAmino Acid Sequence

The SCW nucleotide sequences are used to generate variant nucleotidesequences having the nucleotide sequence of the open reading frame withabout 70%, 75%, 80%, 85%, 90% and 95% nucleotide sequence identity whencompared to the starting unaltered ORF nucleotide sequence of thecorresponding SEQ ID NO. These functional variants are generated using astandard codon table. While the nucleotide sequence of the variants arealtered, the amino acid sequence encoded by the open reading frames donot change. These variants are associated with component traits thatdetermine biomass production and quality. The ones that show associationare then used as markers to select for each component traits.

B. Variant Nucleotide Sequences of SCW in the Non-Coding Regions

The SCW nucleotide sequences are used to generate variant nucleotidesequences having the nucleotide sequence of the 5′-untranslated region,3′-untranslated region, or promoter region that is approximately 70%,75%, 80%, 85%, 90% and 95% identical to the original nucleotide sequenceof the corresponding SEQ ID NO. These variants are then associated withnatural variation in the germplasm for component traits related tobiomass production and quality. The associated variants are used asmarker haplotypes to select for the desirable traits.

C. Variant Amino Acid Sequences of SCW Polypeptides

Variant amino acid sequences of the SCW polypeptides are generated. Inthis example, one amino acid is altered. Specifically, the open readingframes are reviewed to determine the appropriate amino acid alteration.The selection of the amino acid to change is made by consulting theprotein alignment (with the other orthologs and other gene familymembers from various species). An amino acid is selected that is deemednot to be under high selection pressure (not highly conserved) and whichis rather easily substituted by an amino acid with similar chemicalcharacteristics (i.e., similar functional side-chain). Using a proteinalignment, an appropriate amino acid can be changed. Once the targetedamino acid is identified, the procedure outlined in the followingsection C is followed. Variants having about 70%, 75%, 80%, 85%, 90% and95% nucleic acid sequence identity are generated using this method.These variants are then associated with natural variation in thegermplasm for component traits related to biomass production andquality. The associated variants are used as marker haplotypes to selectfor the desirable traits.

D. Additional Variant Amino Acid Sequences of SCW Polypeptides

In this example, artificial protein sequences are created having 80%,85%, 90% and 95% identity relative to the reference protein sequence.This latter effort requires identifying conserved and variable regionsfrom an alignment and then the judicious application of an amino acidsubstitutions table. These parts will be discussed in more detail below.

Largely, the determination of which amino acid sequences are altered ismade based on the conserved regions among SCW protein or among the otherSCW polypeptides. Based on the sequence alignment, the various regionsof the SCW polypeptide that can likely be altered are represented inlower case letters, while the conserved regions are represented bycapital letters. It is recognized that conservative substitutions can bemade in the conserved regions below without altering function. Inaddition, one of skill will understand that functional variants of theSCW sequence of the invention can have minor non-conserved amino acidalterations in the conserved domain.

Artificial protein sequences are then created that are different fromthe original in the intervals of 80-85%, 85-90%, 90-95% and 95-100%identity. Midpoints of these intervals are targeted, with liberallatitude of plus or minus 1%, for example. The amino acids substitutionswill be effected by a custom Perl script. The substitution table isprovided below in Table 4.

TABLE 4 Substitution Table Strongly Rank of Similar and Order Optimal toAmino Acid Substitution Change Comment I L, V 1 50:50 substitution L I,V 2 50:50 substitution V I, L 3 50:50 substitution A G 4 G A 5 D E 6 E D7 W Y 8 Y W 9 S T 10 T S 11 K R 12 R K 13 N Q 14 Q N 15 F Y 16 M L 17First methionine cannot change H Na No good substitutes C Na No goodsubstitutes P Na No good substitutes

First, any conserved amino acids in the protein that should not bechanged is identified and “marked off” for insulation from thesubstitution. The start methionine will of course be added to this listautomatically. Next, the changes are made.

H, C and P are not changed in any circumstance. The changes will occurwith isoleucine first, sweeping N-terminal to C-terminal. Then leucine,and so on down the list until the desired target it reached. Interimnumber substitutions can be made so as not to cause reversal of changes.The list is ordered 1-17, so start with as many isoleucine changes asneeded before leucine, and so on down to methionine. Clearly many aminoacids will in this manner not need to be changed. L, I and V willinvolve a 50:50 substitution of the two alternate optimal substitutions.

The variant amino acid sequences are written as output. Perl script isused to calculate the percent identities. Using this procedure, variantsof the SCW polypeptides are generating having about 80%, 85%, 90% and95% amino acid identity to the starting unaltered ORF nucleotidesequence of SEQ ID NOS: 1, 3, 5 and 40-71.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. An isolated polynucleotide selected from the group consisting of: a.a polynucleotide having at least 70% sequence identity, as determined bythe GAP algorithm under default parameters, to the full length sequenceof a polynucleotide selected from the group consisting of SEQ ID NOS: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,77-114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280,282, 284, 286, 288, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318,320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374,376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402,404, 406, 408 and 410-424; wherein the polynucleotide encodes apolypeptide that functions as a modifier of secondary cell walldevelopment; b. a polynucleotide encoding a polypeptide selected fromthe group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265,267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293,295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321,323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349,351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377,379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405,407 and 409; and c. a polynucleotide selected from the group consistingof SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,67, 69, 71, 73, 77-114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272,274, 276, 278, 280, 282, 284, 286, 288, 300, 302, 304, 306, 308, 310,312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366,368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394,396, 398, 400, 402, 404, 406, 408 and 410-424; and d. A polynucleotidewhich is complementary to the polynucleotide of (a), (b) or (c).
 2. Arecombinant expression cassette, comprising the polynucleotide of claim1, wherein the polynucleotide is operably linked, in sense or anti-senseorientation, to a promoter.
 3. A host cell comprising the expressioncassette of claim
 2. 4. A transgenic plant comprising the recombinantexpression cassette of claim
 2. 5. The transgenic plant of claim 4,wherein said plant is a monocot.
 6. The transgenic plant of claim 4,wherein said plant is a dicot.
 7. The transgenic plant of claim 4,wherein said plant is selected from the group consisting of: maize,soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice,barley, millet, peanut and cocoa.
 8. A transgenic seed from thetransgenic plant of claim
 4. 9. A method of modulating secondary cellwall development in plants, comprising: a. introducing into a plant cella recombinant expression cassette comprising the polynucleotide of claim1 operably linked to a promoter; and b. culturing the plant under plantcell growing conditions; wherein the secondary cell wall development insaid plant cell is modulated.
 10. The method of claim 9, wherein theplant cell is from a plant selected from the group consisting of: maize,soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice,barley, millet, peanut and cocoa.
 11. A method of modulating the wholeplant or secondary cell wall development in a plant, comprising: a.introducing into a plant cell a recombinant expression cassettecomprising the polynucleotide of claim 1 operably linked to a promoter;b. culturing the plant cell under plant cell growing conditions; and c.regenerating a plant form said plant cell; wherein the secondary cellwall development in said plant is modulated.
 12. The method of claim 11,wherein the plant is selected from the group consisting of: maize,soybean, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet,peanut, and cocoa.
 13. A product derived from the method of processingof transgenic plant tissues expressing an isolated polynucleotideencoding an SCW gene, the method comprising: a. transforming a plantcell with a recombinant expression cassette comprising a polynucleotidehaving at least 90% sequence identity to the full length sequence of apolynucleotide selected from the group consisting of SEQ ID NO: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 77-114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254,256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282,284, 286, 288, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348,350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376,378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404,406, 408 and 410-424, operably linked to a promoter; and b. culturingthe transformed plant cell under plant cell growing conditions; whereinthe growth in said transformed plant cell is modulated; c. growing theplant cell under plant-forming conditions to express the polynucleotidein the plant tissue; and d. processing the plant tissue to obtain aproduct.
 14. A product according to claim 13, wherein the polynucleotidefurther encodes a polypeptide selected from the group consisting of SEQID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221,223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249,251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277,279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305,307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333,335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361,363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389,391, 393, 395, 397, 399, 401, 403, 405, 407 and
 409. 15. The transgenicplant of claim 13, wherein the plant is a monocot.
 16. The transgenicplant of claim 13, wherein the plant is selected from the groupconsisting of: maize, soybean, sunflower, sorghum, canola, wheat,alfalfa, cotton, rice, barley, and millet.
 17. A product according toclaim 13, which improves stalk strength of a plant by overexpression ofthe polynucleotide.
 18. A product according to claim 13, which increasesyield by increasing biomass.
 19. A product according to claim 13, whichis a constituent of ethanol.
 20. The method of claim 9, where said planthas improved canopy shape.
 21. The method of claim 9, where said planthas increased photosynthetic capacity in leaf tissue.
 22. The method ofclaim 9, where said plant has improved stalk strength.
 23. The method ofclaim 9, where said plant has improved plant standibility.
 24. Themethod of claim 9, where said plant has altered vascular bundlestructure or number.
 25. The method of claim 9, where said plant hasincreased root biomass.
 26. The method of claim 9, where said plant hasenhanced root growth.
 27. The method of claim 9, where said plant hasmodulated shoot development.
 28. The method of claim 9, where said planthas modulated leaf development.
 29. The method of claim 9, where saidplant has improved silage quality and digestibility.